Modified release orally administered amino acid formulations

ABSTRACT

Methods and formulations of modified release amino acids are provided for the treatment or management of diseases defined by impaired amino acid metabolism, with improved pharmacokinetics, metabolism and utilization.

FIELD OF THE INVENTION

This invention pertains to orally administered formulations and dosageforms of amino acids with improved dosing accuracy, improvedpalatability, and improved pharmacokinetics for optimum therapeuticeffect.

BACKGROUND

Amino acids are necessary building blocks in the synthesis of proteinsby the human body, derived principally from dietary sources of proteins.The human body uses nineteen separate L-configuration amino acids tosynthesize proteins. Some of these amino acids are referred to as“essential amino acids” because the body cannot independently synthesizethem. Other amino acids are not “essential” because the body cansynthesize them.

The digestion of dietary proteins to amino acids starts in the stomach,thanks to the combined activity of pepsinogen, pepsine and hydrochloricacid leading to oligopeptides. The digestion continues in the duodenumafter transit from the stomach through the pylorus, where the combinedactivity of biliary secretion, bicarbonates and pancreatic enzymes isable to produce individual amino acids. Montgomery R. et al., 1988;Baracos, 2004. The liberated amino acids are then absorbed from thesmall intestine into the portal vein according to active stereospecifictransport processes specific for amino acids. The secretion into theintestine of amino acids is a concomitant mechanism. Dipeptides andtripeptides can also be absorbed from the small intestine, but they tooare eventually hydrolyzed into amino acids in the enterocyte. Theseprocesses are largely pH dependent. When food is ingested, the stomachsecretes hydrochloric acid and the pH decreases to around 2. After thepyloric valve, the pH progressively increases from 4 to approximately 7as a consequence of alkaline biliary secretion. A buffer system operatesto prevent pH values larger than approximately 7.5-7.8.

The time required to digest dietary proteins is the sum of the timespent in the gastric emptying phase and the time required for hydrolysisin the small intestine. The time needed for both of these processes canvary depending on food intake, protein intake, illness, and concomitantdrug treatments, with food intake being the most common determiningfactor. Amidon et al., 1995 reports a gastric emptying time ofapproximately 3.5 hours after a high fat meal, approximately 1.5 hoursafter an average meal, and approximately 10 to 20 minutes after 250 mlof water. Keohane et al., 1985 reports the time required to hydrolyzeamino acids in the small intestine, on average, is approximately threehours, and that this time varies for individual proteins and aminoacids. This hydrolysis starts to occur immediately after gastricemptying, after transit through the pyloric valve, and lasts for severalhours in the first tract of the small intestine, namely the jejunum.

Amino acids are commonly administered as supplements to the diet tofacilitate protein synthesis, particularly where increases in musclemass are intended. Specially formulated combinations of amino acids arealso administered to support the nutritional health of individuals withspecial dietary intake limitations and requirements. In phenylketonuria,for example, the body is unable to metabolize phenylalanine to tyrosinedue to the lack of necessary enzymes. The phenylalanine insteaddecomposes to several toxic by-products. Montgomery et al., 1988;Waisbren et al., 2007.

Phenylalanine is an essential amino acid that is normally metabolized totyrosine by the enzyme phenylalanine hydroxylase. In phenylketonuriathis enzyme is absent or not functioning properly, and the phenylalanineis decarboxylated to various compounds, three of them being toxic:phenylpyruvate, phenyl lactate and phenyl acetate. Because of thismetabolic inhibition, the dietary intake of phenylalanine must bereduced while providing the amino acid tyrosine with the diet as it isindispensable. Ney D. M. et al., 2014; Dioguardi, 2011; Waisbren et al.,2007.

An effective management strategy for phenylketonuria consists ofspecially formulated amino acid formulations that omit phenylalaninewhile supplementing tyrosine. Because phenylalanine cannot be removedfrom most dietary sources of protein, these amino acid supplements willusually take the place of dietary proteins, which are eliminated fromthe diet entirely. Vliet Van D. et al., 2014. These patients can,however, also be treated with a carefully controlled diet of proteinsthat have very little phenylalanine. Gropper S. S. et al., 1991.

Examples of amino acid formulations sold for the management ofphenylketonuria include formulations sold as Antifen™, Nutricia XP2™(Maxamaid) and Milupa PKU2™ (Secunda). Nutricia XP2™ and Milupa PKU2™also contain vitamins and minerals. The following Table A gives theformulations of these products, based on about 100 g of amino acids ineach formulation, omitting the vitamin and mineral content.

TABLE A Antifen ™ ¹ Nutricia XP2 ™ ² Milupa PKU2 ™ ³ Component g g gAlanine 7.60 4.05 4.03 Arginine 7.83 7.20 3.32 Aspartic Acid 11.30 6.709.48 Cystine 1.17 2.66 2.25 Glutamic Acid — — 19.92 Glutamine 12.50 4.94— Glycine 4.80 6.35 2.25 Histidine 2.73 4.11 2.25 Isoleucine 3.23 6.355.69 Leucine 7.20 10.86 9.37 Lysine 8.19 7.37 6.64 Methionine 2.14 1.732.25 Proline 4.89 7.70 9.01 Serine 8.10 4.66 4.98 Taurine 0.25 1.17 —Threonine 3.80 5.33 4.51 Tryptophan 1.25 2.14 1.66 Tyrosine 7.50 9.645.57 Valine 5.40 6.97 6.64 Carnitine 0.11 0.06 0.18 ¹http://www.dmfmetabolic.it/wp-content/uploads/2013/10/AntifenIntegrale.pdf² http://www.nutricia.it/prodotti/xp2-maxamaid/ ³http://www.nutricia.it/prodotti/pku-2-secunda/

Other conditions for which specially formulated amino acid preparationsare manufactured and administered, using the same strategy to provideall essential and non-essential amino acids but omitting amino acidsthat the body is unable to metabolize, include tyrosinemia, leucinosis,methylmalonic acidemia, homocistinuria, hyperglycinemia, isovalericacidemia, propionic acidemia, and glutaric acidemia (types II, IIA orIIB).

Free amino acids potentially present numerous disadvantages overnaturally occurring proteins, due to the rapid absorption of the aminoacids, imposing a higher dietary acid load, particularly when higherdoses are administered.

In addition the taste and the odor and taste of some commercial aminoacid formulations is not good, which makes them extremely difficult formany people to ingest on a regular basis, particularly infants andchildren. In addition, these formulations are released immediately wheningested, leading to a very different absorption pattern than whenproteins are ingested.

One object of the present invention is to limit the amino acidcatabolism caused when free amino acids are ingested and overwhelm thebody's metabolic capacity.

Another object of the present invention is to improve the taste of aminoacid formulations when orally ingested.

Still another object of the present invention is to provide uniquedosage forms of amino acids that can be eaten directly without the needfor reconstitution.

Yet another object of the present invention is to provide formulationsof amino acids with modified release properties, that more closely mimicthe absorption pattern of proteins when orally ingested.

SUMMARY OF THE INVENTION

Modified release amino acid formulations have been developed thatovercome the foregoing drawbacks. The formulations are more palatableand, most importantly, mimic the release profile of amino acids fromnaturally derived food sources. In particular, the formulations employ arelease retarding mechanism that modifies the release of amino acidsfrom the formulation to mimic the release rate of amino acids fromnaturally occurring proteins.

In the limited trials done to date, several individual amino acids andcombinations of amino acids, when modified to achieve specific in vitrorelease criteria, have demonstrated statistically superior improvementsin plasma concentrations of amino acids over comparable free amino acidformulations. Thus, in a first principal embodiment, the inventionprovides an orally administered amino acid formulation comprising one ormore modified release amino acids, wherein the formulation comprising 2g of the modified release amino acids releases no more than x % of themodified release amino acids in 30 minutes of dissolution testingperformed in a <711> USP 39 NF 34, paddle apparatus, at 37° C., in 450or 500 mL, 0.1 N hydrochloric acid (pH 1.2), paddle speed 50 rpm,wherein x % is 90%, 80%, 70%, 60%, 50% or even 40%.

In an in vivo animal bioavailability study, it has unexpectedly beendiscovered that isoleucine, leucine, tyrosine, and valine, when modifiedfor release as described herein, consistently have a maximumconcentration in blood that is significantly less than the maximumconcentration produced by a comparable free amino acid formulation.Thus, in a second principal embodiment the invention provides an orallyadministered amino acid formulation comprising a modified release aminoacid selected from arginine, isoleucine, leucine, tyrosine and valine,wherein the formulation comprising 2 g of the modified release aminoacids releases no more than x % of the modified release amino acids in30 minutes of dissolution testing performed in a <711> USP 39 NF 34,paddle apparatus, at 37° C., in 450 or 500 mL, 0.1 N hydrochloric acid(pH 1.2), paddle speed 50 rpm, wherein x % is 90%, 80%, 70%, 60%, 50%,or even 40%.

Another principal embodiment relates to a particular grouping of aminoacids—the branched chain amino acids—and the unexpected discovery thatthe branched chain amino acids, in aggregate, when modified for releaseas described herein, produce a significantly lower C_(max) and higherC_(last) than the C_(max) and C_(last) produced by a comparable freeamino acid formulation. Thus, in another principal embodiment theinvention provides an orally administered amino acid formulationcomprising one or more modified release branched chain amino acids,wherein the formulation comprising 2 g of the modified branched chainrelease amino acids releases no more than x % of the modified releaseamino acids in 30 minutes of dissolution testing performed in a <711>USP 39 NF 34, paddle apparatus, at 37° C., in 450 or 500 mL, 0.1 Nhydrochloric acid (pH 1.2), paddle speed 50 rpm, wherein x % is 90%,80%, 70%, 60%, 50% or even 40%.

Yet another principal embodiment relates to another particular groupingof amino acids—essential amino acids—which, when modified for release asdescribed herein, unexpectedly produce a significantly lower C_(max) andsignificantly higher C_(last) when compared to a comparable free aminoacid formulation. Thus, in still another principal embodiment, theinvention provides an orally administered amino acid formulationcomprising one or more modified release essential amino acids, whereinthe formulation comprising 2 g of the modified release essential aminoacids releases no more than x % of the modified release amino acids in30 minutes of dissolution testing performed in a <711> USP 39 NF 34,paddle apparatus, at 37° C., in 450 or 500 mL, 0.1 N hydrochloric acid(pH 1.2), paddle speed 50 rpm, wherein x % is 90%, 80%, 70%, 60%, 50% oreven 40%.

Still another principal embodiment relates to another grouping of aminoacids—large neutral amino acids (excluding Phenylalanine, Tyrosine andMethionine)—which, when modified for release as described herein,unexpectedly produce a significantly lower C_(max) and significantlyhigher C_(last) when compared to a comparable free amino acidformulation. Thus, in still another principal embodiment, the inventionprovides an orally administered amino acid formulation comprising one ormore modified release large neutral amino acids, wherein the formulationcomprising 2 g of the modified release large neutral amino acidsreleases no more than x % of the modified release amino acids in 30minutes of dissolution testing performed in a <711> USP 39 NF 34, paddleapparatus, at 37° C., in 450 or 500 mL, 0.1 N hydrochloric acid (pH1.2), paddle speed 50 rpm, wherein x % is 90%, 80%, 70%, 60%, 50% oreven 40%.

In a seventh principal embodiment the invention provides an orallyadministered amino acid formulation comprising a combination of modifiedrelease non-essential amino acids wherein the formulation comprising 2 gof the modified release amino acids releases no more than x % of themodified release amino acids in 30 minutes of dissolution testingperformed in a <711> USP 39 NF 34, paddle apparatus, at 37° C., in 450or 500 mL, 0.1 N hydrochloric acid (pH 1.2), paddle speed 50 rpm,wherein x % is 90%, 80%, 70%, 60%, 50% or even 40%.

The formulations are preferably in the form of edible granules that canbe mixed with other foods such as yogurt, or large chewable tablets thatcannot be swallowed whole, but must be eaten like a biscuit in severalbites. The quantity of amino acids in the unit dose formulations willdepend on the metabolic demands of the individual being treatedgenerally depending on the individual's age, sex, weight, and physicalactivities, and whether the individual suffers from a metabolic disorderlimiting his or her intake of one or more amino acids, as described ingreater detail herein.

The formulation may comprise one, all, or any combination of essentialand non-essential amino acids, and can be specifically designed for anyapplication where amino acid supplementation is desired. Theformulations are particularly well suited for the treatment of peoplewith amino acid processing disorders such as phenylketonuria,tyrosinemia, leucinosis, methylmalonic acidemia, homocystinuria,hyperglycinemia, isovaleric acidemia, propionic acidemia, and glutamicacidemia.

The modified release can be accomplished by ingredients referred toherein variously as release retarding agents, means for preferentiallyreleasing the amino acids in the jejunum, etc. These agents are capableof causing a modified release of amino acids from the formulation wheningested or when tested in dissolution media. Alternatively, they arecapable of extending or delaying the release of amino acids so that someof the amino acids are not released until later in the digestiveprocess, such as after the formulation has reached the higher pHenvironment of the small intestine. These agents are even capable, in asuitably designed formulation, of releasing a subset of amino acids inthe formulation at a different release rate than other amino acids. Theformulations are preferably released based on their modified releasepattern when tested at a fixed pH value.

Other aspects of the present invention involve the use of theformulations to treat or manage a disease that requires close managementof amino acid intake, commonly due to an impaired amino acid metabolism.Thus, in another embodiment, the invention provides a method of treatinga metabolic disorder selected from the group consisting ofphenylketonuria, tyrosinemia, leucinosis, methylmalonic acidemia,homocystinuria, hyperglycinemia, isovaleric acidemia, propionicacidemia, and glutamic acidemia, in a patient in need thereof,comprising orally administering to the patient an amino acid formulationof the present invention.

Yet another embodiment provides methods of making the formulations ofthe present invention, and in this embodiment the invention provides amethod of making a formulation of the present invention comprising: (a)providing a first mixture comprising a single or plurality of aminoacids; (b) contacting said mixture with a wetting agent or solvent (e.g.an alcohol and/or an organic solvent and/or water) and a binder to forma wet mixture; (c) optionally passing said wet mixture through a sieveto form a uniform wet granulate; (d) drying said uniform wet granulateto form a dry granulate; (e) optionally passing said dry granulatethrough a sieve to form a uniform dry granulate, and (f) optionallycoating the dry granulate with one or more coating agents to provide amodified release to said dry granulate. Suitable coating agents include,for example, cellulose polymers, fatty acids, waxes and other coatingagent able to provide a modified release to said dry granulate. Suitablecoating techniques include, for example, fluid bed coating and meltgranulation. The dry granulates can be incorporated into anyconventional dosage form but preferably are used as the dosage form,without further modification, where they can easily be mixed with otheringredients such as flavors and inactive excipients or additives, oractive ingredients such as vitamins, minerals and carbohydrates.Alternatively, the dry granulates can be compressed into a chewabletablet.

Additional advantages of the invention are set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

DESCRIPTION OF THE FIGURES

This patent application file contains at least one drawing executed incolor. Copies of this patent application with color drawings will beprovided by the Office upon request and payment of the necessary fee.

FIG. 1 tabulates the 19 amino acids used by the human body to synthesizeproteins, and classifies them according to structure and properties,namely aliphatic, aromatic, acid, basic, sulphurated and pK_(a).

FIG. 2 tabulates essential amino acids, i.e. those amino acids that arenot produced in the body through biochemical synthesis, but must beingested with the diet, as reported by Montgomery et al., 1988.

FIG. 3 is a graphical depiction of the release profile over time ofuncoated tryptophan made according to the method of Example 7, using twodifferent paddle speeds, as compared to tryptophan formulated inaccordance with the present invention.

FIG. 4 is a graphical depiction of the release profile of tryptophanover time from the formulation of Example 8 using a dissolution media atpH 1.2 and a paddle speed of 50 rpm.

FIG. 5 is a graphical depiction of the release profile of tryptophanover time from the formulation of Example 9 when subjected to theexperimental conditions described in Example 8 but using the basketmethod instead of the paddle.

FIG. 6 is a graphical depiction of the ponderal release profiles of aformulation containing the amino acids mixture over time from theformulation of Example 10, when subjected to the experimental conditionsdescribed in Example 10.

FIG. 7 is a graphical depiction of the ponderal dissolution profile ofthe free amino acid formulation described in Example 11, Table 10b,tested according to the methods described in Example 11.

FIG. 8 plots the dissolution profile for the 18 separate amino acidsfrom APR AA mix (bkT037/71)

FIG. 9 is a graphical depiction of the ponderal dissolution profile offour separate ethylcellulose coated modified release amino acidformulations, made and tested according to the methods and formulationreported in Example 11 and Table 10d and 10e.

FIG. 10 plots the dissolution profile for the 18 separate amino acidsfrom APR Batch 2 (bkT037/72-2), Table 10f, when tested according to themethods reported in Example 11.

FIG. 11 plots the dissolution profile for the 18 separate amino acidsfrom APR Batch 4 (bkT037/72-4), Table 10g, when tested according to themethods reported in Example 11.

FIG. 12 plots the aggregate amino acid ponderal dissolution profile fromfour separate ethylcellulose/glyceryl dibehenate coated modified releaseamino acid formulations, manufactured and tested as described in Example12, having the compositions described in Table 11a.

FIG. 13 plots the dissolution profile of 18 separate amino acids fromAPR Batch 5 (bkT037/73-5) (Table 11c), when tested according to themethods reported in Example 12.

FIG. 14 plots the dissolution profile of 18 separate amino acids fromAPR Batch 7 (bkT037/72-7) (Table 11d), when tested according to themethods reported in Example 12.

FIG. 15 depicts the aggregate ponderal amino acid dissolution profilesof four separate test formulations of the current inventions (APR Batch2/bkT037/72-2, APR Batch 4/bkT037/72-4, APR Batch 5/bkT037/73-5, APRBatch 7/bkT037/72-7) and free amino acids mixture bkT037/71,manufactured and tested as described in Examples 11 and 12.

FIG. 16 reports the mean plasma concentrations in pigs of 14 amino acidsover time, on an aggregate basis, from two separate modified releaseamino acid formulations of the present invention, one free amino acidformulation, and casein, as described in Example 14.

FIG. 17 reports the mean plasma concentrations in pigs of the essentialamino acids over time, on an aggregate basis, from two separate modifiedrelease amino acid formulations of the present invention, one free aminoacid formulation, and casein, as described in Example 14.

FIG. 18 reports the mean plasma concentrations in pigs of the branchedchain amino acids over time, on an aggregate basis, from two separatemodified release amino acid formulations of the present invention, onefree amino acid formulation, and casein, as described in Example 14.

FIG. 19 reports the mean plasma concentrations in pigs of the largeneutral chain amino acids over time, on an aggregate basis, from twoseparate modified release amino acid formulations of the presentinvention, one free amino acid formulation, and casein, as described inExample 14.

DETAILED DESCRIPTION Definitions

When the singular forms “a,” “an” and “the” or like terms are usedherein, they will be understood to include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “ahydrocarbon” includes mixtures of two or more such hydrocarbons, and thelike. The word “or” or like terms as used herein means any one member ofa particular list and also includes any combination of members of thatlist.

When used herein the term “about” or “ca.” will compensate forvariability allowed for in the pharmaceutical industry and inherent inpharmaceutical products, such as differences in product strength andbioavailability due to manufacturing variations and time-induced productdegradation. The term allows for any variation which in the practice ofpharmaceuticals would allow the product being evaluated to be consideredpharmaceutically equivalent or bioequivalent, or both if the contextrequires, to the recited strength of a claimed product. It will beunderstood that all numeric values expressed in this document can beprefaced by the term “about.”

The term “independently” is used to mean that the particular elementsmodified by a set of potential variables may or may not be defined bythe same variable. Thus, for example, if a composition comprises z % ofelement A and z % of element B, and z can independently be 10% or 20%,the composition can comprise 10% of element A and 10% of element B, or10% of element A and 20% of element B, etc.

The term “amino acid” refers to any naturally occurring amino acidcapable of participating in the synthesis of peptides and proteins. Forease of drafting, the amino acid will frequently be written without itsstereo-configuration, although it will be understood that the amino acidshould be present as its naturally occurring stereoisomer.

In the formulations of the present invention, amino acids can be presentas the free base, as the hydrochloride salt, or as another suitablesalt. The term essential amino acid refers to any amino acid that thebody is incapable of making itself, which must be derived from anexternal source. Essential amino acids that can be included in theformulations of the present invention include L-Isoleucine, L-Leucine,L-Lysine, L-Threonine, L-Tryptophan, L-Valine, L-Methionine,L-Phenylalanine, L-Histidine and L-Arginine or any of theirpharmaceutically acceptable salts (these last two are consideredessential only for infants although, for simplification, they arereferred to as “essential amino acids herein”).

The term non-essential amino acid refers to any amino acid other than anessential amino acid, and thus includes for example L-Alanine,L-Aspartic Acid, L-Cystine, L-Glutamine and/or L-Glutamic acid, Glycine,L-Proline, L-Serine, Taurine, L-Tyrosine, and L-Carnitine or any oftheir pharmaceutically acceptable salts. Any of these amino acids can bepresent, as the free base, the hydrochloride salt, or any other suitablesalt.

The term “branched chain amino acids” refers to valine, isoleucine, andleucine, or any of their pharmaceutically acceptable salts.

The term “large neutral amino acids” in one embodiment refers totryptophan, threonine, valine, isoleucine, leucine, and histidine, orany of their pharmaceutically acceptable salts. In another embodimentthe term also includes phenylalanine, methionine and tyrosine.

The term “formulation” refers to a finished or semi-finished combinationof pharmaceutical or medical food or food ingredients, including bothactive ingredients and inactive excipients or additives. The term refersto in-process formulations, finished formulations, and formulationspackaged as a final unit dose.

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises,” means “including but not limited to,” and is not intendedto exclude, for example, other additives, components, integers or steps.

The terms “treating” and “treatment,” when used herein, refer to themedical management of a patient with the intent to cure, ameliorate,stabilize, or prevent a disease, pathological condition, or disorder.This term includes active treatment, that is, treatment directedspecifically toward the improvement of a disease, pathologicalcondition, or disorder, and also includes causal treatment, that is,treatment directed toward removal of the cause of the associateddisease, pathological condition, or disorder. In addition, this termincludes palliative treatment, that is, treatment designed for therelief of symptoms rather than the curing of the disease, pathologicalcondition, or disorder; preventative treatment, that is, treatmentdirected to minimizing or partially or completely inhibiting thedevelopment of the associated disease, pathological condition, ordisorder; and supportive treatment, that is, treatment employed tosupplement another specific therapy directed toward the improvement ofthe associated disease, pathological condition, or disorder. The terminclude also dietary management of a patient who, because of a specificdisease or medical condition, has limited or impaired capacity toingest, digest, adsorb or metabolize foodstuffs or nutrients or who hasother special medically determined nutrient requirements, the dietarymanagement of which cannot be achieved by the modification of the normaldiet alone.

The term “modified release” refers to any pharmaceutical formulation inwhich the release rate is intentional altered to achieve a desiredtherapeutic or pharmacokinetic response. The term thus includes extendedrelease formulations, in which the release of the drug is extended overtime, or a release rate that is independent of the pH of the surroundingenvironment. The term also includes delayed release formulations, wherethe release of active ingredient from the formulation (or a portionthereof) is delayed to occur after the initial ingestion. A delayedrelease formulation is typically designed so that release occurspredominantly once the formulation reaches the small intestine.

As used herein, “therapeutically effective amount” refers to an amountsufficient to elicit the desired biological response. Thetherapeutically effective amount or dose will depend on the age, sex andweight of the patient, and the current medical condition of the patient.The skilled artisan will be able to determine appropriate dosagesdepending on these and other factors in addition to the presentdisclosure. When used to refer to nutrients such as amino acids,minerals, vitamins, etc., the term refers to that amount generallyrecognized as necessary to support the human body's metabolic needs.

“Pharmaceutically acceptable” means that which is useful in preparing apharmaceutical composition that is generally safe, non-toxic and neitherbiologically nor otherwise undesirable and includes that which isacceptable for veterinary use as well as human medical food, food andpharmaceutical use. “Pharmaceutically acceptable salts” means salts thatare pharmaceutically or medical food or food acceptable, as definedabove, and which possess the desired pharmacological or nutritiveactivity.

When ranges are expressed herein by specifying alternative upper andlower limits of the range, it will be understood that the endpoints canbe combined in any manner that is mathematically feasible. Thus, forexample, a range of from 50 or 80 to 100 or 70 can alternatively beexpressed as a series of ranges of from 50 to 100, from 50 to 70, andfrom 80 to 100. When a series of upper bounds and lower bounds arerelated using the phase and/or, it will be understood that the upperbounds can be unlimited by the lower bonds or combined with the lowerbounds, and vice versa. Thus, for example, a range of greater than 40%and/or less than 80% includes ranges of greater than 40%, less than 80%,and greater than 40% but less than 80%.

When a specific testing methodology is given from the United StatesPharmacopoeia or other industry-accepted compendium herein, it will beunderstood that the test method can be modified according to usual andcustomary practices without altering the basic physical principals orend-results based on the conventional skill of the skilled artisan, andthe examples provided herein, as, for example, may be needed to testdifferent dosage forms.

When percentages are given herein, it will be understood that thepercentages are weight percent, and that proportions are based onweight, unless otherwise stated to the contrary.

Discussion

The invention is defined in terms of principal embodiments andsubembodiments, and it will be understood that the principal embodimentscan be combined to define other principal embodiments, that thesubembodiments can be combined to define additional subembodiments, andthat the subembodiments and combinations of subembodiments can becombined with all of the principal embodiments to define furtherembodiments of the present invention. The ability to combine embodimentsand subembodiments is limited only by what is mathematically orphysically impossible.

In a first principal embodiment the invention provides an orallyadministered amino acid formulation comprising one or more modifiedrelease amino acids wherein the formulation comprising 2 g of themodified release amino acids releases no more than x % of the modifiedrelease amino acids in 30 minutes of dissolution testing performed in a<711> USP 39 NF 34, paddle apparatus, at 37° C., in 450 or 500 mL, 0.1 Nhydrochloric acid (pH 1.2), paddle speed 50 rpm, wherein x % is 90%,80%, 70%, 60%, 50% or even 40%.

In a second principal embodiment the invention provides an orallyadministered amino acid formulation comprising a modified release aminoacid selected from arginine, isoleucine, leucine, tyrosine and valine,wherein the formulation comprising 2 g of the modified release aminoacids releases no more than x % of the modified release amino acids in30 minutes of dissolution testing performed in a <711> USP 39 NF 34,paddle apparatus, at 37° C., in 450 or 500 mL, 0.1 N hydrochloric acid(pH 1.2), paddle speed 50 rpm, wherein x % is 90%, 80%, 70%, 60%, 50%,or even 40%.

In a third principal embodiment the invention provides an orallyadministered amino acid formulation comprising modified release branchedchain amino acids, wherein the formulation comprising 2 g of themodified release amino acids releases no more than x % of the modifiedrelease amino acids in 30 minutes of dissolution testing performed in a<711> USP 39 NF 34, paddle apparatus, at 37° C., in 450 or 500 mL, 0.1 Nhydrochloric acid (pH 1.2), paddle speed 50 rpm, wherein x % is 90%,80%, 70%, 60%, 50% or even 40%. In a preferred embodiment theformulation includes modified release valine, isoleucine and leucine.

In a fourth principal embodiment, the invention provides an orallyadministered amino acid formulation comprising a combination of modifiedrelease essential amino acids wherein the formulation comprising 2 g ofthe modified release amino acids releases no more than x % of themodified release amino acids in 30 minutes of dissolution testingperformed in a <711> USP 39 NF 34, paddle apparatus, at 37° C., in 450or 500 mL, 0.1 N hydrochloric acid (pH 1.2), paddle speed 50 rpm,wherein x % is 90%, 80%, 70%, 60%, 50% or even 40%. In a preferredembodiment the modified release amino acids include Arginine, Histidine,Isoleucine, Leucine, Threonine, Lysine and Tryptophan.

In a fifth principal embodiment, the invention provides an orallyadministered amino acid formulation comprising modified release largeneutral amino acids, wherein the formulation comprising 2 g of themodified release large neutral amino acids releases no more than x % ofthe modified release amino acids in 30 minutes of dissolution testingperformed in a <711> USP 39 NF 34, paddle apparatus, at 37° C., in 450or 500 mL, 0.1 N hydrochloric acid (pH 1.2), paddle speed 50 rpm,wherein x % is 90%, 80%, 70%, 60%, 50% or even 40%. In a preferredembodiment the formulation includes modified release tryptophan,threonine, valine, isoleucine, leucine, and histidine.

In a sixth principal embodiment the invention provides a method oftreating a metabolic disorder selected from the group consisting ofphenylketonuria, tyrosinemia, leucinosis, methylmalonic acidemia,homocystinuria, hyperglycinemia, isovaleric acidemia, propionicacidemia, and glutamic acidemia, in a patient in need thereof,comprising orally administering to the patient a formulation of thepresent invention defined by any of the principal embodiments or subembodiments.

In a seventh principal embodiment the invention provides an orallyadministered amino acid formulation comprising a combination of modifiedrelease non-essential amino acids wherein the formulation comprising 2 gof the modified release amino acids releases no more than x % of themodified release amino acids in 30 minutes of dissolution testingperformed in a <711> USP 39 NF 34, paddle apparatus, at 37° C., in 450or 500 mL, 0.1 N hydrochloric acid (pH 1.2), paddle speed 50 rpm,wherein x % is 90%, 80%, 70%, 60%, 50% or even 40%.

In various subembodiments, the formulation may comprise one, all, or anycombination of essential and non-essential amino acids, and can bespecifically designed for any application where amino acidsupplementation is desired. The formulations are particularly wellsuited for the treatment of people with amino acid processing disorderssuch as phenylketonuria, tyrosinemia, leucinosis, methylmalonicacidemia, homocystinuria, hyperglycinemia, isovaleric acidemia,propionic acidemia, and glutamic acidemia. Other more particularsubembodiments are described more specifically in Tables B-D. It will beunderstood, however, that some flexibility should be incorporated inthese formulation to account, for example, for the amino acidrequirements of infants and children, which could cause some of theamino acid to fall out of the recited proportions.

Table B presents relative weight parts of nineteen amino acids, for usegenerally as modified release amino acids in formulations intended foramino acid supplementation, as well as phenylketonuria and tyrosinemia.Column A presents preferred ranges of suitable proportions of aminoacids, while column B presents more preferred range of suitableproportions. Table B only describes the relative proportions of aminoacids in the formulation. Thus, other ingredients can be included. Theaggregate rates of amino acid release from any of the formulationsdescribed in Table B are preferably as recited in the first principalembodiment.

TABLE B General Phenylketonuria Tyrosinemia A B A B A B L-Alanine2.0-12.0 2.0-7.0 2.0-12.0 2.0-7.0 2.0-12.0 3.5-5.0 L-Arginine 3.0-10.53.0-8.2 3.0-10.5 3.0-8.0 3.0-10.5 7.0-8.0 L-Aspartic Acid 5.0-10.5 5.0-10.5 5.0-10.5  5.0-10.5 5.0-10.5 6.0-8.0 L-Cystine 1.5-4.0  1.5-4.01.5-4.0  1.0-3.0 1.5-4.0  2.5-3.5 L-Glutamic 7.0-25.0 10.0-22.0 7.0-25.011.0-22.0 7.0-25.0  8.0-12.0 Acid + Glutamine Glycine 3.5-15.0 4.0-8.03.5-15.0 4.0-6.0 3.5-15.0 6.0-8.0 L-Histidine 2.0-6.5  2.5-5.0 2.0-6.5 2.0-3.5 2.0-6.5  4.0-5.0 L-Isoleucine 2.0-8.5  4.0-7.0 2.0-8.5  4.5-6.52.0-8.5  6.0-8.0 L-Leucine 8.0-15.0  8.0-14.0 8.0-15.0  8.0-13.08.0-15.0 10.0-12.5 L-Lysine 4.5-10.5 4.9-9.0 4.5-10.5 4.9-8.0 4.5-10.57.5-9.5 L-Methionine 1.0-3.0  1.0-3.0 1.0-3.0  1.0-3.0 1.0-3.0  1.5-2.5L-Phenylalanine 4.0-7.5  4.5-7.0 0.0 0.0 0.0 0.0 L-Proline 3.5-15.0 4.0-10.0 3.5-15.0 5.0-8.2 3.5-15.0 7.5-9.0 L-Serine 2.0-8.5  3.0-7.02.0-8.5  3.0-7.0 2.0-8.5  4.5-6.0 L-Threonine 4.0-7.5  4.0-6.5 4.0-7.5 4.0-6.0 4.0-7.5  5.0-6.5 L-Tryptophan 1.0-4.0  1.5-3.0 1.0-4.0  1.5-3.01.0-4.0  1.5-3.0 L-Tyrosine 2.5-14.0  6.5-12.0 2.5-14.0  6.5-12.0 0.00.0 L-Valine 2.5-10.0 4.0-8.0 2.5-10.0 4.0-8.0 2.5-10.0 6.5-8.0L-Carnitine 0.0-0.2  0.05-0.2  0.0-0.2  0.05-0.2  0.0-0.2  0.0-0.2Taurine 0.05-1.0  0.1-0.7 0.05-1.0  0.2-0.5 0.05-1.0  0.05-0.5 

Further embodiments are defined when the first principal embodimentincludes as modified release amino acids three or more, five or more, 10or more, or 15 or more, or even all of the amino acids encompassed bythe general formulations of Table B (defined by Column A or B) in theproportions recited in columns A or B.

Further embodiments are defined when the first principal embodimentincludes as modified release amino acids the essential amino acids, thebranched chain amino acids, or the large neutral amino acids encompassedby the general formulations of Table B (defined by Column A or B) in theproportions recited in columns A or B.

Further embodiments are defined when the first principal embodimentincludes as modified release amino acids three or more, five or more, 10or more, or 15 or more, or even all of the amino acids encompassed bythe phenylketonuria formulations of Table B (defined by Column A or B)in the proportions recited in columns A or B.

Further embodiments are defined when the first principal embodimentincludes as modified release amino acids the essential amino acids, thebranched chain amino acids, or the large neutral amino acids encompassedby the phenylketonuria formulations of Table B (defined by Column A orB) in the proportions recited in columns A or B.

Further embodiments are defined when the first principal embodimentincludes as modified release amino acids three or more, five or more, 10or more, or 15 or more, or even all of the amino acids encompassed bythe tyrosinemia formulations of Table B (defined by Column A or B) inthe proportions recited in columns A or B.

Further embodiments are defined when the first principal embodimentincludes as modified release amino acids the essential amino acids, thebranched chain amino acids, or the large neutral amino acids encompassedby the tyrosinemia formulations of Table B (defined by Column A or B) inthe proportions recited in columns A or B.

Table C presents relative weight parts of nineteen amino acids for useas modified release amino acids in leucinosis, methylmalonic acidemia,propionic acidemia, and glutaric acidemia. Column A presents preferredranges of suitable proportions of amino acids for each of thesemetabolic disorders, while column B presents more preferred range ofsuitable proportions. Table C only describes the relative proportions ofamino acids in the formulation. Thus, other ingredients can be included.The aggregate rates of amino acid release from any of the formulationsdescribed in Table C are preferably as recited in the first principalembodiment.

TABLE C Methylmalonic Acidemia Leucinosis or Propionic Acidemia GlutaricAcidemia A B A B A B L-Alanine  2.0-12.0 4.5-6.0  2.0-12.0  9.0-11.0 2.0-12.0 4.0-5.0 L-Arginine  3.0-10.5  8.5-10.5  3.0-10.5  8.0-10.0 3.0-10.5 7.0-8.5 L-Aspartic Acid  5.0-10.5 7.0-8.5  5.0-10.5 7.0-9.0 5.0-10.5 6.0-7.0 L-Cystine 1.5-4.0 3.0-4.0 1.5-4.0 3.0-4.0 1.5-4.02.5-3.5 L-Glutamic Acid +  7.0-25.0  9.0-13.0  7.0-25.0  9.0-11.0 7.0-25.0  8.0-10.0 Glutamine Glycine  3.5-15.0 7.5-9.5  3.5-15.03.5-4.5  3.5-15.0 6.5-8.0 L-Histidine 2.0-6.5 4.5-6.0 2.0-6.5 5.5-7.02.0-6.5 3.5-5.5 L-Isoleucine 0.0 0.0 0.0-0.5 0.0-0.5 2.0-8.5 6.0-8.5L-Leucine 0.0 0.0 10.0-15.0 12.0-15.0 10.0-15.0 10.5-13.0 L-Lysine 4.5-10.5  8.5-10.5  4.5-10.5  8.0-10.5 0.0 0.0 L-Methionine 1.0-3.01.5-3.0 0.0 0.0 1.0-3.0 1.5-2.5 L-Phenylalanine 4.0-7.5 5.5-7.0 4.0-7.55.5-7.5 4.0-7.5 4.5-6.0 L-Proline  3.5-15.0  9.5-11.0  3.5-15.0 3.5-5.0 3.5-15.0 7.5-9.0 L-Serine 2.0-8.5 5.5-7.5 2.0-8.5 5.0-6.5 2.0-8.54.5-6.0 L-Threonine 4.0-7.5 6.5-8.0 0.0 0.0 4.0-7.5 5.0-6.5 L-Tryptophan1.0-4.0 2.5-3.5 1.0-4.0 2.0-3.5 0.0-0.5 0.0-0.5 L-Tyrosine  2.5-14.02.5-7.0  2.5-14.0 5.0-7.0  2.5-14.0 4.5-6.0 L-Valine 0.0 0.0 0.0 0.0 2.5-10.0 6.5-8.0 L-Carnitine 0.0-0.2 0.02-0.12 0.0-0.2  0.0-0.150.0-0.2 0.0-0.2 Taurine 0.05-1.0  0.05-1.0  0.05-1.0  0.05-1.0 0.05-1.0  0.5-0.5

Further embodiments are defined when the first principal embodimentincludes as modified release amino acids three or more, five or more, 10or more, or 15 or more, or even all of the amino acids encompassed bythe leucinosis formulations of Table C (defined by Column A or B) in theproportions recited in columns A or B.

Further embodiments are defined when the first principal embodimentincludes as modified release amino acids the essential amino acids, thebranched chain amino acids, or the large neutral amino acids encompassedby the leucinosis formulations of Table C (defined by Column A or B) inthe proportions recited in columns A or B.

Further embodiments are defined when the first principal embodimentincludes as modified release amino acids three or more, five or more, 10or more, or 15 or more, or even all of the amino acids encompassed bythe methylmalonic acidemia formulations of Table C (defined by Column Aor B) in the proportions recited in columns A or B.

Further embodiments are defined when the first principal embodimentincludes as modified release amino acids the essential amino acids, thebranched chain amino acids, or the large neutral amino acids encompassedby the methylmalonic acidemia formulations of Table C (defined by ColumnA or B) in the proportions recited in columns A or B.

Further embodiments are defined when the first principal embodimentincludes as modified release amino acids three or more, five or more, 10or more, or 15 or more, or even all of the amino acids encompassed bythe propionic acidemia formulations of Table C (defined by Column A orB) in the proportions recited in columns A or B.

Further embodiments are defined when the first principal embodimentincludes as modified release amino acids the essential amino acids, thebranched chain amino acids, or the large neutral amino acids encompassedby the propionic acidemia formulations of Table C (defined by Column Aor B) in the proportions recited in columns A or B.

Further embodiments are defined when the first principal embodimentincludes as modified release amino acids three or more, five or more, 10or more, or 15 or more, or even all of the amino acids encompassed bythe glutaric acidemia formulations of Table C (defined by Column A or B)in the proportions recited in columns A or B.

Further embodiments are defined when the first principal embodimentincludes as modified release amino acids the essential amino acids, thebranched chain amino acids, or the large neutral amino acids encompassedby the glutaric acidemia formulations of Table C (defined by Column A orB) in the proportions recited in columns A or B.

Table D presents relative weight parts of nineteen amino acids for useas modified release amino acids in isovaleric acidemia, homocystinuria,and hyperglycinemia. Column A presents preferred ranges of suitableproportions of amino acids for each of these metabolic disorders, whilecolumn B presents more preferred ranges of suitable proportions. Table Donly describes the relative proportions of amino acids in theformulation. Thus, other ingredients can be included. The aggregaterates of amino acid release from any of the formulations described inTable D are preferably as recited in the first principal embodiment.

TABLE D Isovaleric Acidemia Homcystinuria Hyperglycinemia A B A B A BL-Alanine  2.0-12.0 4.0-6.0  2.0-12.0 3.0-5.0  2.0-12.0 3.5-5.0L-Arginine  3.0-10.5 7.0-9.0  3.0-10.5 6.5-8.0  3.0-10.5 6.5-8.5L-Aspartic Acid 5.0-10  6.0-7.0  5.0-10.0 5.0-7.0  5.0-10.0 6.0-8.0L-Cystine 1.5-4.0 2.5-3.5 1.5-4.0 2.0-3.0 1.5-4.0 2.0-3.5 L-GlutamicAcid +  7.0-25.0 8.0-9.5  7.0-25.0 7.5-9.0  7.0-25.0  7.0-10.0 GlutamineGlycine  3.5-15.0 13.0-16.0  3.5-15.0 5.5-7.0 0.0 0.0 L-Histidine2.0-6.5 4.0-5.0 2.0-6.5 3.5-4.5 2.0-6.5 4.0-5.0 L-Isoleucine 2.0-8.52.0-3.5 2.0-8.5 5.5-7.0 2.0-8.5 6.0-8.5 L-Leucine 0.0 0.0 10.0-15.010.0-12.0 10.0-15.0 10.0-12.5 L-Lysine  4.5-10.5 6.0-7.5  4.5-10.57.0-8.0  4.5-10.5 7.0-8.5 L-Methionine 1.0-3.0 1.5-2.5 0.0 0.0 1.0-3.01.0-2.5 L-Phenylalanine 4.0-7.5 5.0-6.0 4.0-7.5 4.0-5.5 4.0-7.5 4.5-6.0L-Proline  3.5-15.0 13.0-15.0  3.5-15.0 7.0-8.5  3.5-15.0 7.0-9.0L-Serine 2.0-8.5 4.0-5.0 2.5-8.5 5.0-8.5 2.5-8.5 4.5-5.5 L-Threonine4.0-7.5 4.0-5.5 4.0-7.5 5.0-6.0 4.0-7.5 5.0-6.5 L-Tryptophan 1.0-4.01.5-2.5 1.0-4.0 1.5-2.5 1.0-4.0 2.0-3.0 L-Tyrosine  2.5-14.0 4.5-6.0 2.5-14.0 4.5-6.0  2.5-14.0 4.0-6.0 L-Valine  2.5-10.0 2.5-4.0 2.5-8.06.0-8.0  2.5-10.0 6.0-8.0 L-Carnitine 0.0-0.2  0.0-0.15 0.0-0.2 0.0-0.10.0-0.2 0.05-0.2  Taurine 0.05-1.0  0.05-0.5  0.05-1.0  0.5-0.50.05-1.0  0.1-0.5

Further embodiments are defined when the first principal embodimentincludes as modified release amino acids three or more, five or more, 10or more, or 15 or more, or even all of the amino acids encompassed bythe isovaleric acidemia formulations of Table D (defined by Column A orB) in the proportions recited in columns A or B.

Further embodiments are defined when the first principal embodimentincludes as modified release amino acids the essential amino acids, thebranched chain amino acids, or the large neutral amino acids encompassedby the isovaleric acidemia formulations of Table D (defined by Column Aor B) in the proportions recited in columns A or B.

Further embodiments are defined when the first principal embodimentincludes as modified release amino acids three or more, five or more, 10or more, or 15 or more, or even all of the amino acids encompassed bythe homocystinuria formulations of Table D (defined by Column A or B) inthe proportions recited in columns A or B.

Further embodiments are defined when the first principal embodimentincludes as modified release amino acids the essential amino acids, thebranched chain amino acids, or the large neutral amino acids encompassedby the homocystinuria formulations of Table D (defined by Column A or B)in the proportions recited in columns A or B.

Further embodiments are defined when the first principal embodimentincludes as modified release amino acids three or more, five or more, 10or more, or 15 or more, or even all of the amino acids encompassed bythe hyperglycinemia formulations of Table D (defined by Column A or B)in the proportions recited in columns A or B.

Further embodiments are defined when the first principal embodimentincludes as modified release amino acids the essential amino acids, thebranched chain amino acids, or the large neutral amino acids encompassedby the hyperglycinemia formulations of Table D (defined by Column A orB) in the proportions recited in columns A or B.

Additional subembodiments are defined when the third principalembodiment is limited by the relative proportions of branched chainamino acids recited in table E1-E3.

TABLE E1 General Phenylketonuria Tyrosinemia A B A B A B L-Isoleucine2.0-8.5  4.0-7.0 2.0-8.5  4.5-6.5 2.0-8.5  6.0-8.0 L-Leucine 8.0-15.0 8.0-14.0 8.0-15.0  8.0-13.0 8.0-15.0 10.0-12.5 L-Valine 2.5-10.04.0-8.0 2.5-10.0 4.0-8.0 2.5-10.0 6.5-8.0

TABLE E2 Methylmalonic Acidemia Leucinosis or Propionic AcidemiaGlutaric Acidemia A B A B A B L-Isoleucine 0.0 0.0 0.0-0.5 0.0-0.52.0-8.5 6.0-8.5 L-Leucine 0.0 0.0 10.0-15.0 12.0-15.0 10.0-15.010.5-13.0 L-Valine 0.0 0.0 0.0 0.0  2.5-10.0 6.5-8.0

TABLE E3 Isovaleric Acidemia Homcystinuria Hyperglycinemia A B A B A BL-Isoleucine 2.0-8.5  2.0-3.5 2.0-8.5 5.5-7.0 2.0-8.5 6.0-8.5 L-Leucine0.0 0.0 10.0-15.0 10.0-12.0 10.0-15.0 10.0-12.5 L-Valine 2.5-10.02.5-4.0 2.5-8.0 6.0-8.0  2.5-10.0 6.0-8.0

Additional subembodiments are defined when the fourth principalembodiment is limited by the relative proportions of essential aminoacids (Arginine, Histidine, Isoleucine, Leucine, Lysine, Threonine,Tryptophan and Valine) recited in tables F1-F3.

TABLE F1 General Phenylketonuria Tyrosinemia A B A B A B L-Arginine3.0-10.5 3.0-8.2 3.0-10.5 3.0-8.0 3.0-10.5 7.0-8.0 L-Histidine 2.0-6.5 2.5-5.0 2.0-6.5  2.0-3.5 2.0-6.5  4.0-5.0 L-Isoleucine 2.0-8.5  4.0-7.02.0-8.5  4.5-6.5 2.0-8.5  6.0-8.0 L-Leucine 8.0-15.0  8.0-14.0 8.0-15.0 8.0-13.0 8.0-15.0 10.0-12.5 L-Lysine 4.5-10.5 4.9-9.0 4.5-10.5 4.9-8.0  4.5-10.5 7.5-9.5 L-Threonine 4.0-7.5  4.0-6.5 4.0-7.5  4.0-6.04.0-7.5  5.0-6.5 L-Tryptophan 1.0-4.0  1.5-3.0 1.0-4.0  1.5-3.0 1.0-4.0 1.5-3.0 L-Valine 2.5-10.0 4.0-8.0 2.5-10.0 4.0-8.0 2.5-10.0 6.5-8.0

TABLE F2 Methylmalonic Acidemia Leucinosis or Propionic AcidemiaGlutaric Acidemia A B A B A B L-Arginine  3.0-10.5  8.5-10.5  3.0-10.5 8.0-10.0  3.0-10.5 7.0-8.5 L-Histidine 2.0-6.5 4.5-6.0 2.0-6.5 5.5-7.02.0-6.5 3.5-5.5 L-Isoleucine 0.0 0.0 0.0-0.5 0.0-0.5 2.0-8.5 6.0-8.5L-Leucine 0.0 0.0 10.0-15.0 12.0-15.0 10.0-15.0 10.5-13.0 L-Lysine 4.5-10.5  8.5-10.5  4.5-10.5  8.0-10.5 0.0 0.0 L-Threonine 4.0-7.56.5-8.0 0.0 0.0 4.0-7.5 5.0-6.5 L-Tryptophan 1.0-4.0 2.5-3.5 1.0-4.02.0-3.5 0.0-0.5 0.0-0.5 L-Valine 0.0 0.0 0.0 0.0  2.5-10.0 6.5-8.0

TABLE F3 Isovaleric Acidemia Homcystinuria Hyperglycinemia A B A B A BL-Arginine  3.0-10.5 7.0-9.0  3.0-10.5 6.5-8.0  3.0-10.5 6.5-8.5L-Histidine 2.0-6.5 4.0-5.0 2.0-6.5 3.5-4.5 2.0-6.5 4.0-5.0 L-Isoleucine2.0-8.5 2.0-3.5 2.0-8.5 5.5-7.0 2.0-8.5 6.0-8.5 L-Leucine 0.0 0.010.0-15.0 10.0-12.0 10.0-15.0 10.0-12.5 L-Lysine  4.5-10.5 6.0-7.5 4.5-10.5 7.0-8.0  4.5-10.5 7.0-8.5 L-Threonine 4.0-7.5 4.0-5.5 4.0-7.55.0-6.0 4.0-7.5 5.0-6.5 L-Tryptophan 1.0-4.0 1.5-2.5 1.0-4.0 1.5-2.51.0-4.0 2.0-3.0 L-Valine  2.5-10.0 2.5-4.0 2.5-8.0 6.0-8.0  2.5-10.06.0-8.0

Additional subembodiments are defined when the fifth principalembodiment is limited by the relative proportions of large neutral aminoacids recited in table G1-G3.

TABLE G1 General Phenylketonuria Tyrosinemia A B A B A B L-Histidine2.0-6.5 2.5-5.0 2.0-6.5 2.0-3.5 2.0-6.5 4.0-5.0 L-Isoleucine 2.0-8.54.0-7.0 2.0-8.5 4.5-6.5 2.0-8.5 6.0-8.0 L-Leucine  8.0-15.0  8.0-14.0 8.0-15.0  8.0-13.0  8.0-15.0 10.0-12.5 L-Threonine 4.0-7.5 4.0-6.54.0-7.5 4.0-6.0 4.0-7.5 5.0-6.5 L-Tryptophan 1.0-4.0 1.5-3.0 1.0-4.01.5-3.0 1.0-4.0 1.5-3.0 L-Valine  2.5-10.0 4.0-8.0  2.5-10.0 4.0-8.0 2.5-10.0 6.5-8.0

TABLE G2 Methylmalonic Acidemia Leucinosis or Propionic AcidemiaGlutaric Acidemia A B A B A B L-Histidine 2.0-6.5 4.5-6.0 2.0-6.55.5-7.0 2.0-6.5 3.5-5.5 L-Isoleucine 0.0 0.0 0.0-0.5 0.0-0.5 2.0-8.56.0-8.5 L-Leucine 0.0 0.0 10.0-15.0 12.0-15.0 10.0-15.0 10.5-13.0L-Threonine 4.0-7.5 6.5-8.0 0.0 0.0 4.0-7.5 5.0-6.5 L-Tryptophan 1.0-4.02.5-3.5 1.0-4.0 2.0-3.5 0.0-0.5 0.0-0.5 L-Valine 0.0 0.0 0.0 0.0 2.5-10.0 6.5-8.0

TABLE G3 Isovaleric Acidemia Homcystinuria Hyperglycinemia A B A B A BL-Histidine 2.0-6.5 4.0-5.0 2.0-6.5 3.5-4.5 2.0-6.5 4.0-5.0 L-Isoleucine2.0-8.5 2.0-3.5 2.0-8.5 5.5-7.0 2.0-8.5 6.0-8.5 L-Leucine 0.0 0.010.0-15.0 10.0-12.0 10.0-15.0 10.0-12.5 L-Threonine 4.0-7.5 4.0-5.54.0-7.5 5.0-6.0 4.0-7.5 5.0-6.5 L-Tryptophan 1.0-4.0 1.5-2.5 1.0-4.01.5-2.5 1.0-4.0 2.0-3.0 L-Valine  2.5-10.0 2.5-4.0 2.5-8.0 6.0-8.0 2.5-10.0 6.0-8.0

In another subembodiment for the treatment of phenylketonuria, definedwithout regard to the proportions of amino acids, the formulation of thepresent invention comprises three or more of, five or more of, or allof, arginine, histidine, isoleucine, leucine, lysine, methionine,threonine, tryptophan, and valine, or one or more pharmaceuticallyacceptable salts thereof, as modified release essential amino acids.

In another subembodiment for the treatment of phenylketonuria, againdefined without regard to the proportions of amino acids, theformulations of the present invention comprise three or more of, five ormore of, or all of, arginine, histidine, isoleucine, leucine, lysine,methionine, threonine, tryptophan, and valine, or one or morepharmaceutically acceptable salts thereof, as modified release essentialamino acids; and three or more of, five or more of, or all of, alanine,aspartic acid, carnitine, cystine, glutamine and/or glutamic acid,glycine, proline, serine, taurine, and tyrosine, or one or morepharmaceutically acceptable salts thereof, as modified releasenon-essential amino acids.

In one particular subembodiment, which is suitable for the treatment ofany of the metabolic disorders described herein, defined without regardto the proportions of ingredients, the formulation comprises arginineand histidine, or one or more pharmaceutically acceptable salts thereof,as modified release essential amino acids; and alanine, aspartic acid,carnitine, cystine, glutamine and/or glutamic acid, proline, serine, andtaurine, or one or more acceptable salts thereof, as modified releasenon-essential amino acids.

In a particularly preferred embodiment, the formulation is provided as asingle dose, unit dosage form, comprising from 1 to 40 grams of aminoacids, wherein the formulation is therapeutically effective to meet theamino acid dietary needs of a human patient when administered three tofour times per day. I.e., the formulation is adequate to meet thepatient's dietary needs for the amino acids which are actually presentin the formulation.

In another principal embodiment, the invention provides a method ofmaking the formulation defined by any of the principal embodiments orsubembodiments of the present invention, comprising: (a) providing afirst mixture comprising a single or plurality of amino acids; (b)contacting the mixture with a wetting agent and a binder to form a wetmixture; (c) optionally passing the wet mixture through a sieve to forma uniform wet granulate; (d) drying the uniform wet granulate to form adry granulate; (e) optionally passing the dry granulate through a sieveto form a uniform dry granulate; and (f) optionally coating the uniformdry granulate with a modified release composition.

The method is particularly well suited for mixing large number of aminoacids, and could be adapted further by preparing more than one granulatewith different mixtures of amino acids, and combining the differentgranulates to make the final formulation. Thus, in a preferredembodiment of the method of manufacture, the first mixture couldcomprise only one type of amino acid, or as many as 6 amino acids,further comprising repeating steps (a)-(d) and optionally (e) and (f)with a second different mixture of amino acids comprising only one aminoacid, or as many as 6 amino acids to form a second uniform drygranulate, and combining the uniform dry granulates to make theformulation. In one embodiment the granulates are compressed to form achewable tablet. In another embodiment, after the granulate mixture isformed, it is further processed by, for example the addition offlavorings or nutrients.

The formulations can also be defined based on the percentage of aminoacids in the formulation. Thus, for example, the invention furtherprovides formulations comprising greater than 20 wt %, 70 wt % or 90 wt% amino acids, wherein (a) said amino acids comprise from about 10 wt %to about 80 wt %, from about 20 wt % to about 60 wt %, from about 30 toabout 50%, or from about 35 to about 45% essential amino acids; and (b)said essential amino acids comprise all or any combination ofL-Isoleucine, L-Leucine, L-Lysine, L-Threonine, L-Tryptophan, L-Valine,L-Methionine, L-Histidine and L-Arginine (the last two ones areconsidered essential only for infants).

Alternatively, the modified release formulation can be defined based onthe ratio of essential to non-essential amino acids in the formulation.Thus, in various embodiments the invention provides a modified releaseorally administered amino acid formulation comprising greater than 20 wt%, 50%, 70 wt % or 90 wt % amino acids, wherein (a) said amino acidscomprise essential and non-essential amino acids in a weight ratio offrom about 10:90, 20:80. 30:70, 40:60, 50:50, 60:40, 70:30 80:20 toabout 90:10, 80:20, 70:30, 60:40, 50:50. 40:60, 30:70, or 20:80; and (b)said essential amino acids comprise all or any combination ofL-Isoleucine, L-Leucine, L-Lysine, L-Threonine, L-Tryptophan, L-Valine,L-Methionine, L-Histidine and L-Arginine (the last two ones areconsidered essential only for infants).

The formulation preferably includes one or more excipients or additivescapable of controlling the release of all the amino acids in theformulation, or a subset of the amino acids. These excipients oradditives are referred to herein generally as “release retardingexcipients or additives,” and are capable of providing an extendedrelease profile, a delayed release profile, a combination of immediaterelease and extended release or delayed release profiles. In oneembodiment the excipients or additives are delayed release excipients oradditives and are referred to as “means for preferentially releasingsaid amino acids at a pH between 4 and 7” or “means for preferentiallyreleasing said amino acids in the jejunum when orally administered.” Insuch case the excipient is typically present in an amount effective tocause preferential release of said amino acids from said formulation ata pH between 4 and 7.

The term “preferential release” does not exclude the possibility thatsome of the excipient will release amino acids in a lower pHenvironment, or even that the rate of release in a lower pH environmentwill equal that which occurs in a higher pH environment. The importantpoint is that the excipient reduces the total quantity of amino acidsthat would otherwise be released in the low pH environment of thestomach versus an immediate release dosage form. Thus, for example, theexcipient can yield a slower release profile over time as theformulation travels through the GI tract than would otherwise occur withan immediate release formulation, or as modeled in a suitable in vitroexperiment. Alternatively, the excipient can retard the release untilthe formulation reaches the higher pH environment of the jejunum.

Release modifying excipients or additives/coating agents are well knownto those of skill in the art and include, for example, ethylcellulose,cellulose acetate, vinyl acetate/vinyl chloride copolymers,acrylate/methacrylate copolymers, polyethylene oxide, hydroxypropylmethylcellulose, carrageenan, alginic acid and salts thereof,hydroxyethyl cellulose, hydroxypropyl cellulose, karaya gum, acacia gum,tragacanth gum, locust bean gum, guar gum, xanthan gum, sodiumcarboxymethyl cellulose, methyl cellulose, beeswax, carnauba wax, cetylalcohol, hydrogenated vegetable oils, stearyl alcohol, acrylic acidcopolymers, carrageenan, pectin, one sodium carboxymethyl cellulose,mono and diglycerides of fatty acids, fatty acids and their esters orderivatives including glyceryl behenates and glyceryl dipalmitostearate,starches and their derivatives such as maize acetate.

Formulations

These compositions of the present invention can be prepared in a mannerwell known in the pharmaceutical and food supplement art, and can beadministered by a variety of routes depending upon whether local orsystemic treatment is desired. The most preferred route ofadministration is oral. The compositions will typically contain, inaddition to the amino acids, one or more acceptable carriers (excipientsor additives). In making the compositions of the invention, the activeingredient is typically mixed with an excipient, diluted by an excipientor enclosed within such a carrier in the form of, for example, acapsule, sachet, paper, or other container. When the excipient serves asa diluent, it can be a solid, semi-solid, or liquid material, which actsas a vehicle, carrier or medium for the active ingredient. Thus, thecompositions can be in the form of tablets, pills (including soft andhard gelatin capsules), powders, granulates, microspheres, lozenges,sachets (i.e. packaged powders or granulates or microspheres) sachets,elixirs, suspensions, emulsions, solutions, and syrups, containing, forexample, up to 90% by weight amino acids.

Using this technology is possible to design various amino acid products,in which all or only a select portion of amino acids has the modifiedrelease profile. For example it is possible to design a formulationwhere:

-   -   all or only a portion of the amino acids in the formulation are        formulated for modified release. I.e., greater than 20%, 40%,        60% or 80%, and/or less than 80%, 60%, 40%, or 20% of the amino        acids are formulated for modified release.    -   all or only a portion of the essential amino acids in the        formulation are formulated for modified release. I.e., greater        than 20%, 40%, 60% or 80%, and/or less than 80%, 60%, 40%, or        20% of the essential amino acids are formulated for modified        release.    -   none of the essential amino acids are formulated for modified        release.    -   all or only a portion of the non-essential amino acids in the        formulation are formulated for modified release. I.e., greater        than 20%, 40%, 60% or 80%, and/or less than 80%, 60%, 40%, or        20% of the non-essential amino acids are formulated for modified        release.    -   none of the non-essential amino acids are formulated for        modified release.    -   any combination of the foregoing strategies.

For example, using the formulation strategies described in thisapplication, it is possible to formulate amino acid dosage forms, sothat only the essential amino acids are formulated for modified release,and the non-essential amino acids are not. Alternatively, it is possibleto formulate amino acid dosage forms so that only the non-essentialamino acids are formulated for modified release, and the essential aminoacids are not. Alternatively, depending on the results of releasetesting for a particular formulation, it might be best to keep some ofthe amino acids, such as tyrosine, as immediate release amino acids.

Various strategies are available to control the release of amino acidsfrom the formulation, depending on the desired pharmacokinetic profile.These strategies will typically involve non-pH dependent excipients oradditives, so that the formulation releases amino acids independently ofits location in the gastrointestinal tract, or pH-dependent excipientsor additives, to delay the release of amino acids until the formulationreaches the higher pH environment of the small intestine.

Non-pH-dependent sustained release agents/coatings which may be includedin the composition include, but are not limited to, ethylcellulose,cellulose acetate, vinyl acetate/vinyl chloride copolymers,acrylate/methacrylate copolymers, polyethylene oxide, hydroxypropylmethylcellulose, carrageenan, alginic acid and salts thereof,hydroxyethyl cellulose, hydroxypropyl cellulose, xanthan gum, karayagum, acacia gum, tragacanth gum, locust bean gum, guar gum, sodiumcarboxymethyl cellulose, methyl cellulose, beeswax, carnauba wax, cetylalcohol, hydrogenated vegetable oils, stearyl alcohol, fatty acids andtheir esters or derivatives including for example glyceryl behenates andglyceryl dipalmitostearate, xanthan gum, starches, and derivatives ofstarches such as maize acetate.

In general, the at least one non-pH-dependent modified release agent ispresent in the composition in an amount of from about 1 wt % or 5 wt %to about 50 wt % or 25 wt %, preferably from about 1 wt % to about 30 wt%. It is to be understood, however, that the scope of the presentinvention is not to be limited to any particular non-pH-dependentsustained release agents.

pH-dependent agents that increase the rate of release of the amino acidsfrom the formulation at a pH in excess of 5.5 include, but are notlimited to, polymers that swell at a pH in excess of 5.5, enteric agentsfor coating the amino acid, and agents that increase the solubility ofthe amino acids at a pH greater than 5.5, by maintaining an acidicmicroenvironment in the formulation, e.g., an organic acid. Suitableorganic acids include, for example, formic acid, acetic acid, propionicacid, butyric acid, valeric acid, caproic acid, oxalic acid, lacticacid, malic acid, citric acid, benzoic acid, and carbonic acid. Anycombination of these strategies can be employed to promote the releaseof amino acids at a pH greater than 5.5. The at least one pH-dependentagent is typically present in the composition in an amount of from about0.5 wt. % to about 40 wt. %, preferably from about 1 wt. % to about 20wt. %.

Polymers that swell at a pH in excess of 5.5 include, but are notlimited to, acrylic acid copolymers, methacrylic acid copolymers(including for example those sold under the Eudragit® brand), sodiumalginate, carrageenan, alginic acid, pectin, and sodium carboxymethylcellulose.

Enteric agents include, but are not limited to, cellulose acetatephthalate, hydroxypropyl methylcellulose phthalate, polyvinyl acetatephthalate, methacrylic acid copolymers, cellulose acetate trimellitate,hydroxypropyl methylcellulose acetate, succinate, shellac, and zein.

Agents that increase the solubility of the amino acids at a pH greaterthan 5.5 include, but are not limited to, organic acids. Such organicacids maintain an acidic microenvironment in the tablet, and include,but are not limited to, citric acid, fumaric acid, tartaric acid, adipicacid, glucono delta-lactone, and malic acid.

The compositions of the present invention are typically first preparedby wet granulation, coating and, if a biscuit is desired, subsequentlycompressed into a biscuit dosage form after mixing with other suitableexcipients or additives such as lubricants, anti-adherents anddisintegrating agents. Flavoring agents can be added during thegranulation steps so that they are part of the granules or“intragranular,” or subsequently mixed with the granulates, prior tocompression of the granulates and optionally before being compressedinto a tablet/biscuit.

In the wet granulation method, the at least one amino acid and otheringredients are granulated with a granulating fluid (e.g., isopropylalcohol, ethyl alcohol, trichloromethane or water) in a planetary mixer,high shear mixer, or fluidized bed granulator. Binding agents may becontained in the granulating fluid, or may be in the dry mix. The wetgranules are dried in an oven or fluidized bed dryer, and then sievedthrough a suitable screen to obtain free flowing granules that can bethen be coated with functional excipients or additives able to give amodified or prolonged or delayed release profile.

Bulking agents can be included within the granules or biscuitformulation and include, but are not limited to, microcrystallinecellulose, mannitol, xylitol, dicalcium phosphate, calcium sulfate,starches, lactose, sucrose, dextrose, sorbitol, fructose, and cellulosepowder. When present, the bulking agent may be present in thecomposition in an amount of from about 5 wt. % to about 90 wt. %,preferably from about 10 wt. % to about 50 wt. %.

Disintegrating agents which may be included in the formulations include,but are not limited to, microcrystalline cellulose, starches,crospovidone, sodium starch glycolate, and crosscarmellose sodium. Whenpresent, the disintegrating agent may be present in the composition inan amount of from about 0.5 wt. % to about 30 wt %, preferably fromabout 1 wt. % to about 15 wt. %.

Anti-adherants and glidants which may be employed in the compositioninclude, but are not limited to, talc, corn starch, silicon dioxide,sodium lauryl sulfate, and metallic stearates. When present, theantiadherant or glidant may be present in the composition in an amountof from about 0.2 wt. % to about 15 wt. %, preferably from about 0.5 wt.% to about 5 wt. %.

Lubricants which may be employed in the composition include, but are notlimited to, magnesium stearate, calcium stearate, sodium stearate,stearic acid, sodium stearyl fumarate, hydrogenated cotton seed oil,talc, and waxes, including but not limited to, beeswax, carnuba wax,cetyl alcohol, glyceryl stearate, glyceryl palmitate, glyceryl behenate,hydrogenated vegetable oils, and stearyl alcohol. When present, thelubricant may be present in an amount of from about 0.2 wt. % to about20 wt. %, preferably from about 0.5 wt. % to about 5 wt. %.

Binding agents (“binders”) which may be employed include, but are notlimited to, polyvinyl pyrrollidone, starch, methylcellulose,hydroxypropyl methylcellulose, carboxymethyl cellulose, sucrosesolution, dextrose solution, guar gum, xanthan gum, acacia, tragacanth,locust bean gum and sodium alginate or any other alginic acid salt. Whenpresent, the binding agent may be present in the composition in anamount of from about 0.2 wt. % to about 10 wt. %, preferably from about0.5 wt. % to about 5 wt. %.

Coating agents which may be included in the composition include, but arenot limited to, ethylcellulose, cellulose acetate, vinyl acetate/vinylchloride copolymers, acrylate/methacrylate copolymers, polyethyleneoxide, hydroxypropyl methylcellulose, carrageenan, alginic acid andsalts thereof, hydroxyethyl cellulose, hydroxypropyl cellulose, karayagum, acacia gum, tragacanth gum, locust bean gum, guar gum, sodiumcarboxymethyl cellulose, methyl cellulose, beeswax, carnauba wax, cetylalcohol, hydrogenated vegetable oils, stearyl alcohol, fatty acids andtheir esters or derivatives including, glyceryl behenates and glyceryldipalmitostearate, xanthan gum, starches, and starch derivativesincluding maize acetate.

Taste-Masking Materials

Amino Acids are inherently bitter tasting and in one embodiment of thepresent invention, these bitter Amino Acids are microencapsulated with ataste-masking material. Materials useful for masking the taste ofpharmaceutical formulations include those materials capable ofmicroencapsulating the Amino Acid, thereby protecting the senses fromits bitter taste. Taste-masking materials of the present inventionprovide superior pharmaceutical formulations by e.g., creating a morepalatable pharmaceutical formulation as compared to pharmaceuticalformulations and/or by creating a dosage form requiring less of thetraditional flavoring or taste masking agents.

Taste-masking materials include, e.g., cellulose hydroxypropyl ethers(HPC); low-substituted hydroxypropyl ethers (L-HPC); cellulosehydroxypropyl methyl ethers (HPMC); methylcellulose polymers;Ethylcelluloses (EC) and mixtures thereof; Polyvinyl alcohol (PVA);hydroxyethylcelluloses; carboxymethylcelluloses and salts ofcarboxymethylcelluloses (CMC); polyvinyl alcohol and polyethylene glycolco-polymers; monoglycerides, triglycerides, polyethylene glycols,modified food starch, acrylic polymers and mixtures of acrylic polymerswith cellulose ethers; cellulose acetate phthalate; sepifilms such asmixtures of HPMC and stearic acid, cyclodextrins, and mixtures of thesematerials.

In addition to microencapsulating the Amino Acids with a taste-maskingmaterial or a material that enhances the shelf-life of the formulationas described herein, the pharmaceutical formulations of the presentinvention may also comprise one or more flavoring agents. “Flavoringagents” or “sweeteners” useful in the pharmaceutical formulations of thepresent invention include, e.g., acacia syrup, acesulfame K, alitame,anise, apple, aspartame, banana, Bavarian cream, berry, black currant,butterscotch, calcium citrate, camphor, caramel, cherry, cherry cream,chocolate, cinnamon, bubble gum, citrus, citrus punch, citrus cream,cotton candy, cocoa, cola, cool cherry, cool citrus, cyclamate,dextrose, eucalyptus, eugenol, fructose, fruit punch, ginger,glycyrrhetinate, glycyrrhiza (licorice) syrup, grape, grapefruit, honey,isomalt, lemon, lime, lemon cream, monoammonium glyrrhizinate, maltol,mannitol, maple, marshmallow, menthol, mint cream, mixed berry,neohesperidine DC, neotame, orange, pear, peach, peppermint, peppermintcream, raspberry, root beer, rum, saccharin, safrole, sorbitol,spearmint, spearmint cream, strawberry, strawberry cream, stevia,sucralose, sucrose, sodium saccharin, saccharin, aspartame, neotame,acesulfame potassium, mannitol, talin, xylitol, sucralose, sorbitol,swiss cream, tagatose, tangerine, thaumatin, tutti fruitti, vanilla,walnut, watermelon, wild cherry, wintergreen, xylitol, or anycombination of these flavoring ingredients, e.g., anise-menthol,cherry-anise, cinnamon-orange, cherry-cinnamon, chocolate-mint,honey-lemon, lemon-lime, lemon-mint, menthol-eucalyptus, orange-cream,vanilla-mint, and mixtures thereof. In other embodiments, sodiumchloride is incorporated into the pharmaceutical formulation.

Based on the amino acids and excipients or additives, one of skilled inthe art would be able to determine the best combination of flavors toprovide the optimally flavored product for consumer demand andcompliance. See, e.g., Roy et al., Modifying Bitterness: Mechanism,Ingredients, and Applications (1997).

Methods of Microencapsulation

The amino acid may be microencapsulated by methods known by one ofordinary skill in the art. Such known methods include, e.g., spraydrying processes, spinning disk processes, hot melt processes, spraychilling methods, fluidized bed, electrostatic deposition, centrifugalextrusion, rotational suspension separation, polymerization atliquid-gas or solid-gas interface, pressure extrusion, or sprayingsolvent extraction bath. In addition to these, several chemicaltechniques, e.g., complex coacervation, solvent evaporation,polymer-polymer incompatibility, interfacial polymerization in liquidmedia, in situ polymerization, in-liquid drying, and desolvation inliquid media could also be used. Furthermore, other methods such as drygranulation (i.e., roller compaction and slugging),extrusion/spheronization, or nano particle coating may also be used.

When microencapsulated, the amino acids can be present in particle sizesranging from 1 micron to 1000 microns, 5 microns to 200 microns, or 10microns to 100 microns.

Spray drying is often more readily available for scale-up to acommercial scale. In various embodiments, the material used in thespray-dry encapsulation process is emulsified or dispersed into the corematerial in a concentrated form, e.g., 10-60% solids. In someembodiments of the present invention, the solid loading is between about10-20%, or between about 10-40%, or between about 40-60%. Themicroencapsulation material is, in one embodiment, is emulsified untilabout 1 to 3 μm droplets are obtained. In other embodiments, themicroencapsulation material is emulsified until about 1 to 200 μmdroplets are obtained, or until about 1 to 100 μm droplets are obtained.Once a dispersion of amino acid and encapsulation material are obtained,the emulsion is fed as droplets into the heated chamber of the spraydrier. In some embodiments, the droplets are sprayed into the chamber orspun off a rotating disk. The microspheres are then dried in the heatedchamber and fall to the bottom of the spray drying chamber where theyare harvested.

Coacervation involves microencapsulation of materials such as activepharmaceutical ingredients and involves a three part process of particleor droplet formation, coascerate wall formation, and capsule isolation.This method can produce very small particle size microcapsules (10-70microns).

Extrusion/spheronization is another method that involves wet massing ofactive pharmaceutical ingredients, followed by the extrusion of the wetmass through a perforated plate to produce short cylindrical rods. Theserods are subsequently placed into a rapidly rotating spheronizer toshape the cylindrical rods into uniform spheres. The spheres aresubsequently dried using a fluid bed drier and then coated with afunctional coating using a fluid bed equipped with a Wurster insert andspray nozzle. This method produces smooth, uniform spheres that areideal for receiving a functional coating. Drug loadings as high as 80%are possible (depending on drug characteristics).

Methods of Dry Coating

In addition to microencapsulation, the stability or release time of theamino acids used in the present invention may be increased byalternative methods such as dry coating and nano-particle coating. Drycoating involves the formation of granules of coated amino acid whichare then mixed with other components. Dry granulation is achieved byforming dense compacts which are subsequently milled to a desiredparticle size and then blended with other components of thepharmaceutical composition. Dry granulation and nano-particle coatingcan provide enhanced stability and taste masking characteristics toactive pharmaceutical by diluting and isolating such components in agranulated matrix of compatible ingredients that can enhance the shelflife of amino acid products as well as taste mask the bitterness ifsweetener or flavors are used in coating material.

Typical technique for dry granulation is to use slugging or rollercompaction. During slugging process, the dry powders are compressedusing a conventional tablet machine, or more usually, a large heavy dutyrotary press. The resulting compacts or “slug” are then milled to adesired particle size. Roller compaction is an alternative gentlermethod; the powder mix being squeezed between two rollers to form acompressed sheet. The sheet normally is weak and brittle and breaksimmediately into flakes. These flakes need gentler treatment to breakthem into granules, and this can be usually be achieved by screeningalone. Parikh, D. M., Handbook of Pharmaceutical Granulation Technology,(Marcel Dekker ed. 1997).

Additional Ingredients

Other ingredients can also be incorporated into the formulations of thepresent invention, including vitamins, minerals, fats, fatty acids suchas DHA, EPA and ARA (arachidonic acid), carbohydrates, and othercompounds, preferably in therapeutically effective amounts. Suitableminerals include, for example, calcium, chloride, chromium, copper,fluoride, iodine, iron, magnesium, manganese, molybdenum, phosphorus,potassium, selenium, sulfur, sodium, and zinc. Examples of suitablevitamins include Vitamin A (Retinol, retinyl acetate and palmitate,beta-carotene), Thiamin (vitamin B1), Riboflavin (vitamin B2), Niacin(vitamin B3, nicotinic acid), Panthenoic Acid (vitamin B5), Vitamin B6(pyridoxine), Vitamin B12 (cobalamin), Biotin, Vitamin C (ascorbicacid), Vitamin D (calciferol), Vitamin E (alpha-tocopherol), Folic Acid(folate), and Vitamin K (phylloquinone, menaquinone).

In one embodiment, the formulation comprises one or more, five or more,10 or more, or all of the following nutrients in addition to the aminoacids present in the formulation, in therapeutically effective amounts:choline, inositol, vitamin A, vitamin D, vitamin E, vitamin K, vitaminC, thiamin, riboflavin, niacin, vitamin B6, folate, vitamin B12, biotin,pantothenic acid, potassium, calcium, magnesium, iron, zinc, copper,manganese, selenium, chromium, molybdenum, iodine, sodium, phosphorus,chloride, docosahexaenoic acid, arachidonic acid and lutein.

Of course, it will be understood that any of the foregoing nutrients canbe present in a form well known to workers of ordinary skill in the artthat provides similar nutritive value, such as salts and chelates andesters and other derivatives of the foregoing nutrients. Table Hdescribes an example of various nutrients, effective nutrient doses, andnutrient derivates capable of incorporation into the formulations of thepresent invention. The formulation can include any one or combination ofthese nutrients.

TABLE H over 16 1-3 years 3-16 years years Substance gr/day gr/daygr/day Vitamin vitamin A 0.00040000 0.00065000 0.00072000 vitamin D0.00003200 0.00003250 0.00003600 vitamin E 0.00360000 0.009750000.02070000 vitamin K 0.00008000 0.00032500 0.00045000 vitamin C0.04000000 0.07150000 0.13500000 thiamin 0.00060000 0.001040000.00135000 riboflavin 0.00060000 0.00195000 0.00270000 niacin 0.007200000.01300000 0.01800000 vitamin B6 0.00040000 0.00227500 0.00135000 folate0.00012000 0.00013000 0.00036000 vitamin B12 0.00000600 0.000052000.00005400 biotin 0.00060000 0.00260000 0.00315000 pantothenic acid0.00400000 0.02275000 0.02700000 Mineral potassium 0.16000000 0.780000000.90000000 calcium 0.50000000 0.97500000 0.76500000 magnesium 0.060000000.22750000 0.19800000 iron 0.01100000 0.01625000 0.01350000 zinc0.00320000 0.01040000 0.01710000 copper 0.00060000 0.00084500 0.00090000manganese 0.00040000 0.00182000 0.00252000 selenium 0.000014000.00003250 0.00009000 chromium 0.00000400 0.00003250 0.00006300molybdenum 0.00002000 0.00006500 0.00007200 iodine 0.00008000 0.000110500.00015300 sodium 0.03600000 0.00000000 0.00000000 phosphorus 0.200000000.78000000 0.63000000 chloride 0.08000000 0.00000000 0.00000000 OtherDHA 0.08000000 0.26000000 0.27000000 nutrients docosahexaenoic acid ARAarachidonic 0.15000000 0.00000000 0.00000000 acid Lutein 0.006000000.00000000 0.00000000 coline 0.07000000 0.14950000 0.18000000 inositol0.06000000 0.07800000 0.09000000

Final Formulation

In a particularly preferred embodiment the formulation comprisesgranulates of amino acids coated by one or release modifying excipients,also referred to herein as “coating means for retarding the amino acidrelease rate,” or “coating means for achieving the recited releaserate.” The granulates can be made by wet or dry granulation techniques,as discussed above, but they are preferably made by wet granulation.They are also preferably confined to a particular size range, such as0.1-3 mm, 0.5-2.0 mm, 0.5-1.0 mm, 0.5-2.0 mm, or 1.0-2.0 mm. Each aminoacid can be contained within its own granulate, but the modified releaseamino acids are preferably mixed within the granulates.

The modified release properties are preferably achieved with a suitablerelease modifying coating or coatings applied to the granulate, in anamount of from 1 wt % to 30 wt %, or from 5 wt % to 25 wt % based on theweight of the amino acids. Suitable release retarding excipients for thecoating are described elsewhere in this document, but one preferredcomposition comprises a coating of from 1 wt % to 15 wt %, from 2 wt %to 10 wt %, or from 5 wt % to 7.5 wt % ethylcellulose based on theweight of the amino acids. Another preferred composition comprises afirst coating of ethylcellulose (as described above) and a secondcoating of from 5% to 15% or about 10 wt. % glyceryl dibehenate based onthe weight of the amino acids.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how themethods claimed herein are made and evaluated, and are intended to bepurely exemplary of the invention and are not intended to limit thescope of what the inventors regard as their invention. Efforts have beenmade to ensure accuracy with respect to numbers (e.g., amounts,temperature, etc.) but some errors and deviations should be accountedfor.

Example 1—Exemplary Amino Acid Mixtures for Phenylketonuria

Table 1 lists four separate representative amino acid mixtures for thedietary management of phenylketonuria, on a percentage basis.

TABLE 1 A B C Essential Amino Acids L-Isoleucine 5.11 7.18 5.11L-Leucine 9.18 12.90 9.18 L-Lysine HCL 7.39 10.39 5.91¹ L-Threonine 3.85.34 5.28 L-Tryptophan 1.68 2.36 1.68 L-Valine 6.39 8.98 6.39L-Methionine 2.04 2.86 2.04 L-Histidine HCl 3.04 2.358 3.04¹ L-Arginine6.14 4.764 6.14 Total Ess AA 41.70 54.764 44.77 Non-Essential AminoAcids L-Alanine 6.0 4.665 6.0 L-Aspartic Acid 9.18 7.122 9.20 L-Cystine2.03 1.57 2.03 L-Glutamine 12.49 9.691 12.49 Glycine 4.48 3.47 4.48L-Proline 7.21 5.594 7.21 L-Serine 5.9 4.57 5.9 Taurine 0.300 0.232 0.25L-Tyrosine 7.51 5.82 7.51 L-Carnitine 0.16 0.124 0.16 Total Non-Ess AA58.30 45.236 55.23 Total AA 100 100 100 ¹weights based on the free base

Example 2—Exemplary Powder Formulations and Manufacturing Process

The following table describes formulations of five separate productscontaining 18 amino acids (excluding Methionine) using Plasdone K26/32as a binding agent and a taste masking agent (Products 2, 3, 4, and 5).The reference formulation (Product 1) is without Plasdone K26/32 andwithout flavors.

TABLE 2a Composition of Products 1, 2, 3, 4 and 5 Prod- Prod- Prod-Prod- Prod- uct 1 uct 2 uct 3 uct 4 uct 5 Component g g g g g L-Alanine5.240 5.240 5.240 5.240 5.240 L-Arginine 6.140 6.140 6.140 6.140 6.140L-Aspartic Acid 9.180 9.180 9.180 9.180 9.180 L-Cystine 2.030 2.0302.030 2.030 2.030 L-Glutamine 12.490 12.490 12.490 12.490 12.490 Glycine4.480 4.480 4.480 4.480 4.480 L-Histidine HCl 3.040 3.040 3.040 3.0403.040 L-Isoleucine 5.110 5.110 5.110 5.110 5.110 L-Leucine 9.180 9.1809.180 9.180 9.180 L-Lysine HCL 7.390 7.390 7.390 7.390 7.390 L-Proline7.210 7.210 7.210 7.210 7.210 L-Serine 5.900 5.900 5.900 5.900 5.900Taurine 0.188 0.188 0.188 0.188 0.188 L-Threonine 4.540 4.540 4.5404.540 4.540 L-Tryptophan 1.680 1.680 1.680 1.680 1.680 L-Tyrosine 7.5107.510 7.510 7.510 7.510 L-Valine 6.360 6.360 6.360 6.360 6.360L-Carnitine 0.116 0.116 0.116 0.116 0.116 Sum 97.784 97.784 97.78497.784 97.784 Plasdone K26/32 0.000 1.000 1.000 1.000 1.000 Flavor¹0.000 0.000 1.250 1.400 1.500 ¹Product 1 no Flavor; Product 2 no Flavor;Product 3 Caramel Flavor; Product 4 Vanilla Flavor; Product 5 BananaFlavor

Each of the formulations was manufactured according to the followinggeneral method.

Step 1 Manufacturing Equipment:

-   -   Balances of various types    -   High share mixer: Diosna Laboratory mixer P1/6    -   Sieves    -   Static oven    -   Mixing machine

Mixture of Amino Acids Product 1

Accurately weigh the amino acids and mix for 20 minutes with the mixingmachine.

Granulate Preparation without Flavor Product 2

Accurately weigh the amino acids and mix for 20 minutes with the mixingmachine. Transfer the mix to the Diosna, and mix for 1 minutes at ablade speed of 250 rpm. Wet the mixture with an alcoholic solution ofPlasdone K26/32 (10% in ethanol solution-10 g for 100 g of Ethanol).After one minute and 30 seconds, add the solution and granulate and mixfor three minutes at a blade speed of 250 rpm and a chopper speed of 500rpm. Discharge the granulate and pass through a sieve with a width of0.8 mm. Dry in a static oven for about four hours at 40° C. Dischargeand pass again through a sieve with a width of 0.8 mm.

Granulate Preparation with Flavor Products 3, 4 and 5

Weigh accurately the amino acids and mix for 20 minutes with the mixingmachine. Transfer the mixture to the Diosna, and mix for 1 minute and 30seconds at a blade speed of 250 rpm. Wet the mixture with an alcoholicsolution of Plasdone K26/32 (10% content in ethanol solution −10 g for100 g of AA) and flavor. The quantity of flavors added to the alcoholicsolution of Plasdone K26/32 are the following:

-   -   1.13 g of 58% content of Caramel in Ethanol (for Product 3)    -   1.13 g of 60% content of Vanilla in Ethanol (for Product 4)    -   1.46 g of 14% content of Banana in Ethanol (for Product 5)        After one minute and 30 seconds mix the solution and granulate        for three minutes at a blade speed of 250 rpm and a chopper        speed of 500 rpm. Discharge the granulate and pass through a        sieve with a width of 0.8 mm. Dry in a static oven for about        four hours at 40° C. Discharge and pass the mixture through a        sieve with a width of 0.8 mm.

Step 2

Preparation of Methionine using a Plasdone K26/32 as binding agent andtaste masking agent plus a very small quantity of Caramel flavor using amanufacturing method similar to the method used for Products 3, 4 and 5reported in step 1.

TABLE 2b Composition of granulate L-Methionine Product 6 Product 6Component g L-Methionine¹ 250.0 Plasdone K26/32² 30 Flavor Caramel³ 3.6Sum 289 ¹100 g of L-Methionine corresponds to 103.2 g of L-Methioninegranulate ²Plasdone K26/32 20% in Ethanol solution: 30 g corresponds to6 g of Plasdone K29/32 ³Caramel in 58% ethanol solution: 3.6 gcorrespond to 2.088 g of pure Caramel

Step 3

Preparation of the final formulation mixing the powder/granulates ofStep 1 plus the granulate of Step 2 in the quantity below reported.

TABLE 2c Composition of final formulation of Products 7, 8, 9, 10 and 11Prod- Prod- Prod- Prod- Prod- uct 7 uct 8 uct 9 uct 10 uct 11 Componentg g g g g L-Alanine 5.240 5.240 5.240 5.240 5.240 L-Arginine 6.140 6.1406.140 6.140 6.140 L-Aspartic Acid 9.180 9.180 9.180 9.180 9.180L-Cystine 2.030 2.030 2.030 2.030 2.030 L-Glutamine 12.490 12.490 12.49012.490 12.490 Glycine 4.480 4.480 4.480 4.480 4.480 L-Histidine HCl3.040 3.040 3.040 3.040 3.040 L-Isoleucine 5.110 5.110 5.110 5.110 5.110L-Leucine 9.180 9.180 9.180 9.180 9.180 L-Lysine HCL 7.390 7.390 7.3907.390 7.390 L-Proline 7.210 7.210 7.210 7.210 7.210 L-Serine 5.900 5.9005.900 5.900 5.900 Taurine 0.188 0.188 0.188 0.188 0.188 L-Threonine4.540 4.540 4.540 4.540 4.540 L-Tryptophan 1.680 1.680 1.680 1.680 1.680L-Tyrosine 7.510 7.510 7.510 7.510 7.510 L-Valine 6.360 6.360 6.3606.360 6.360 L-Carnitine 0.116 0.116 0.116 0.116 0.116 L-Methionine¹2.115 2.115 2.115 2.115 2.115 SumAA 99.899 99.899 99.899 99.899 99.899Plasdone K26/32 0.050 1.050 1.050 1.050 1.050 Flavor² 0.000 0.000 1.2501.400 1.500 ¹2.115 g of granulate L-Methionine in Product 7corresponding to 2.050 g of Methionine ²Product 7 no Flavor (only asmall quantity of caramel deriving from Methionine powder), Product 8 noFlavor (only a small quantity of caramel deriving from Methioninepowder), Product 9 Caramel Flavor, Product 10 Caramel and VanillaFlavor, Product 11 Caramel and Banana Flavor

All the above mentioned Products were manufactured by mixing the powderor granulates of Products 2, 3, 4 and 5 with the granulate of Product 6.

Example 3—Taste Testing

On the final formulations reported in Example 2 (Products 8, 9, 10 and11), the following trial was performed in 6 healthy adults. The resultsreported are average values:

TABLE 3 Results of Taste Testing Reference Marketed Prod- Prod- Prod-Prod- Product uct 8 uct 9 uct 10 uct 11 Product in Yogurt with bananaflavor: ODOR 4.25 4.6 — — — TASTE 3.75 4.6 — — — Persistency (bad tastein 3 2.2 — — — the mouth) Product in Orange juice: ODOR 4 5 — — — TASTE3.25 4.125 — — — Persistency (bad taste in 2.5 1.75 — — — the mouth)Product in pressed Banana ODOR 3.5 5 — — — TASTE 1 4.375 — — —Persistency (bad taste in 2.75 2.25 — — — the mouth) Product dispersedin Water: ODOR 2.4 — 4.3 4.8 4.3 TASTE 0.8 — 3.9 3.5 5 Persistency (badtaste in 4.6 — 2.4 2 1.4 the mouth) Dispersibility 2 4 4 4 4 Legend:Odor/Taste: 0 = bad 5 =good Persistency (bad taste in the mouth): 0 =low persistency 5 = long persistency Dispersability: 0 = bad 5 = good

As the foregoing data shows, the formulations of the present inventionare better than the Reference Marketed Product and better than theformulation without Plasdone K26/32 in terms of dispersability andtaste.

Example 4—Exemplary Biscuit Formulations and Manufacturing Process

Several biscuit formulations were prepared starting with similargranulate Products to those reported in Example 2 (changing slightly theamounts of flavors).

TABLE 4a Product 1 Product 2 Product 3 Component g g g L-Alanine 5.2405.240 5.240 L-Arginine 6.140 6.140 6.140 L-Aspartic Acid 9.180 9.1809.180 L-Cystine 2.030 2.030 2.030 L-Glutamine 12.490 12.490 12.490Glycine 4.480 4.480 4.480 L-Histidine HCl 3.040 3.040 3.040 L-Isoleucine5.110 5.110 5.110 L-Leucine 9.180 9.180 9.180 L-Lysine HCL 7.390 7.3907.390 L-Proline 7.210 7.210 7.210 L-Serine 5.900 5.900 5.900 Taurine0.188 0.188 0.188 L-Threonine 4.540 4.540 4.540 L-Tryptophan 1.680 1.6801.680 L-Tyrosine 7.510 7.510 7.510 L-Valine 6.360 6.360 6.360L-Carnitine 0.116 0.116 0.116 L-Methionine¹ 2.115 2.115 2.115 Sum 99.89999.899 99.899 Plasdone K26/32 1.050 1.050 1.050 Flavor² 0.986 1.02 0.306¹2.115 g of granulate L-Methionine corresponding to 2,050 g ofMethionine ²Product 1 Caramel Flavor, Product 2 Vanilla Flavor, Product3 Banana FlavorTo the above mentioned Products were added saccharose, silicon dioxideand glyceryl dibehenate through a simple mixing procedure. The finalformulations were as follows:

TABLE 4b Product 4¹ Product 5¹ Product 6¹ Component g g g L-Alanine5.240 5.240 5.240 L-Arginine 6.140 6.140 6.140 L-Aspartic Acid 9.1809.180 9.180 L-Cystine 2.030 2.030 2.030 L-Glutamine 12.490 12.490 12.490Glycine 4.480 4.480 4.480 L-Histidine HCl 3.040 3.040 3.040 L-Isoleucine5.110 5.110 5.110 L-Leucine 9.180 9.180 9.180 L-Lysine HCL 7.390 7.3907.390 L-Proline 7.210 7.210 7.210 L-Serine 5.900 5.900 5.900 Taurine0.188 0.188 0.188 L-Threonine 4.540 4.540 4.540 L-Tryptophan 1.680 1.6801.680 L-Tyrosine 7.510 7.510 7.510 L-Valine 6.360 6.360 6.360L-Carnitine 0.116 0.116 0.116 L-Methionine² 2.115 2.115 2.115 Sum 99.89999.899 99.899 Plasdone K26/32 1.050 1.050 1.050 Flavor 0.986 1.02 0.306Saccharose 50.000 50.000 50.000 Glyceryl di-behenate 1.500 1.500 1.500¹Product 4 Caramel Flavor, Product 5 Vanilla Flavor, Product 6 BananaFlavor ²2.115 g of granulate L-Methionine corresponding to 2.050 g ofMethionine

Tablets were prepared using a mono-punch tableting machine with anaverage weight between 5.5 and 6.2 g. To correct some sticking problemsin the punch, a new formulation starting from Product 1 was prepared:

TABLE 4c Product 7 Component g L-Alanine 5.240 L-Arginine 6.140L-Aspartic Acid 9.180 L-Cystine 2.030 L-Glutamine 12.490 Glycine 4.480L-Histidine HCl 3.040 L-Isoleucine 5.110 L-Leucine 9.180 L-Lysine HCL7.390 L-Proline 7.210 L-Serine 5.900 Taurine 0.188 L-Threonine 4.540L-Tryptophan 1.680 L-Tyrosine 7.510 L-Valine 6.360 L-Carnitine 0.116L-Methionine¹ 2.115 Sum 99.899 Polyvinylpyrrolidone 1.050 Flavor Caramel0.986 Comprizuker 50.000 Silicon dioxide 2.500 Glyceryl di-behenate2.500 ¹2.115 g of granulate L-Methionine correspond to 2,050 g ofMethionine

Example 5—Taste Trial of Biscuit Formulation

Products 4, 5 and 6 reported in Example 4 were tested for odor and tasteon 6 healthy adults. The results reported are average values.

TABLE 5 Flavor Flavor Flavor Product 4 Product 5 Product 6 Odor 4.6 3.84.8 Taste 4.4 3.8 3.6 Persistency (bad taste 2.6 3.2 3.2 in the mouth)Legend: Odor-Taste-Vista: 0 = bad 5 = good Persistency (bad taste inmouth): 0 = low persistency 5 = long persistency Dispersability: 0 = bad5 = good

Example 6—Preparation of Modified Release Formulations MixingPreparation

Weigh and sieve each individual amino acid, diluent (if used) and HPMCand mix for 20 minutes in an appropriate mixing machine.

Granulate Preparation without Flavor

Transfer the mix to the Diosna mixer, mixing for 1 minute with bladespeed set at 250 rpm. Wet the mix with an alcoholic solution of EC (15%content). After one minute and 30 seconds add the solution and granulatefor three minutes with a blade speed at 250 rpm and chopper speed at 500rpm. Discharge the granulate and pass through a sieve with a width of0.63 mm. Dry in a static oven for about four hours at 45° C. Dischargeand pass through a sieve with a width of 0.8 mm.

It is also possible to process 4-5 amino acids into granulates at atime, and mix all of the amino acids granulates together to make thefinal product. Some examples of potential formulations are reported inExample 1 Table 1.

Example 7—Modified Release Formulation

The following formula was prepared to determine the quantity ofhydroxypropyl-methylcellulose (HPMC) and ethyl cellulose (EC) needed tomodify the release of tryptophan in a Dissolution Test.

TABLE 6 Composition of Product Component g L-Isoleucine 14 L-Lysine HCL20 L-Threonine 12 L-Tryptophan 4 Mannitol 10 HPMC (Methocel 4KM) 5Solution of EC at 15% in EtOH 19.5¹ ¹corresponding to 3 g of EC

Mixing Preparation

Weigh and sieve the single amino acids, mannitol and HPMC and mix for 20minutes in an appropriate container with the mixing machine.

Granulate Preparation without Flavor

Transfer the mixture to the Diosna mixer, mixing for 1 minute with theblade speed at 250 rpm. Wet the mix with an alcoholic solution of EC(ethyl cellulose) (15% content). After one minute and 30 seconds add thesolution and granulate for three minutes with the blade speed at 250 rpmand the chopper speed at 500 rpm. Discharge the granulate and passthrough a sieve with a width of 0.63 mm. Dry in a static oven for aboutfour hours at 45° C. Discharge and pass through a sieve with a width of0.8 mm.

The formulation was evaluated under the dissolution conditions reportedbelow to determine the release rate of tryptophan from the formulation(pH 1.2 for 2 hours, increased to pH 6.8 after 2 hours) in basketconditions at 50 and 100 rpm versus a simple mixture of uncoated AA. Theresults of the Dissolution Test are reported in FIG. 3. Analysis wasperformed by HPLC.

Dissolution Test Conditions

-   Apparatus: Paddle-   Temperature: 37° C.±5° C.-   Medium: 0.1 N Hydrochloric Acid-   pH adjustment: 0.2 M solution of trisodium phosphate dodecahydrate.    Volume medium: 450 ml 0.1N HCl. After 120 minutes adjust dissolution    medium to pH 6.8±0.05 (according to European Pharmacopoeia 2.9.3    Delayed-release solid dosage forms Method A).-   Speed: 50 rpm or 100 rpm-   Sampling Time: 5 and 15 minutes for uncoated formulation 5, 15, 30,    60, 120, 125, 135, 150, 180 minutes for coated formulation (after    120 minutes the pH of the medium was changed to pH 6.8)-   Vessel concentration: about 90 μg/ml (a fraction of the powder of    the retard formulation remains as surfactant during the dissolution    test)

Example 8—Modified Release Formulation without Mannitol

The following formulation was prepared in accordance with the proceduresset forth in Example 7 and subjected to dissolution testing under theconditions reported below. The release rate of tryptophan from theformulation is depicted in FIG. 4. Analysis was performed by HPLC.

TABLE 7 Composition of the Product Component g L-Isoleucine 56 L-LysineHCL 80 L-Threonine 48 L-Tryptophan 16 HPMC (Methocel 4KM) 20 Solution ofEC at 15% in ETOH 80

Dissolution Test Conditions

-   Apparatus: Paddle-   Temperature: 37° C.±5° C.-   Medium: 0.1 N Hydrochloric Acid-   Volume medium: 450 ml-   Speed: 50 rpm-   Sampling Times: 5′, 15′, 30′, 60′, 120′, 180′-   Vessel concentration: about 90 μg/ml (a fraction of the powder of    the retard formulation remains as surfactant during the dissolution    test)

Example 9—Modified Release Formulation without Mannitol or HPMC(Methocel 4KM)

The following formulation was prepared in accordance with the proceduresset forth in Example 7 and subjected to dissolution testing under theconditions reported below. The release rate of tryptophan from theformulation is depicted in FIG. 5. Analysis was performed by HPLC.

TABLE 8 Composition of the Product Component g L-Isoleucine 56.0L-Lysine 80.0 L-Treonine 48.0 L-Tryptophan 16.0 Ethylcellulose¹ 10.0Total 210 g ¹corresponding to 67 g of an ethanol solution ofEthylcellulose at 15%

Mixing Preparation

Weigh and sieve the single amino acids and mix for 20 minutes in anappropriate container with the mixing machine.

Granulate Preparation without Flavor

Transfer the mixture to the Diosna mixer, mixing for 1 minute with theblade speed at 250 rpm. Wet the mix with an alcoholic solution of EC(ethyl cellulose) (15% content). After one minute and 30 seconds add thesolution and granulate for three minutes with the blade speed at 500 rpmand the chopper speed at 1500 rpm. Discharge the granulate and passthrough a sieve with a width of 0.8 mm. Dry in a static oven for about 5hours and 30 minutes at 45° C. Discharge and pass through a sieve with awidth of 1.4 mm.

The formulation was evaluated under the dissolution conditions reportedbelow to determine the release rate of tryptophan from the formulation(pH 1.2) in basket conditions at 50 rpm. The results of the dissolutiontest are reported in FIG. 5.

Dissolution Test Conditions

-   Apparatus: Basket-   Temperature: 37° C.±5° C.-   Medium: 0.1 N Hydrochloric Acid-   Volume medium: 450 ml-   Speed: 50 rpm-   Sampling Times: 5′, 15′, 30′, 60′, 120′, 180′, 240′, 300′ and 360′-   Vessel concentration: about 90 μg/ml (a fraction of the powder of    the retard formulation remains as surfactant during the dissolution    test)

Example 10—Modified Release Formulation on 19 Amino Acids Coated withEthylcellulose and Glyceryl Dibehenate

The following formulation was prepared in accordance with the proceduresdescribed below.

TABLE 9 Composition of the Product Component Final mix (g) Final mix (%)L-Isoleucine 3.20 4.18 L-Lysine HCl 4.63 6.05¹ L-Treonine 3.31 4.33L-Tryptophan 1.05 1.37 L-Leucine 5.75 7.52 L-Valine 4.00 5.23L-Methionine 1.28 1.67 L-Cystine 1.27 1.66 L-Glutamine 7.82 10.22L-Histidine HCl 2.35 3.07² L-Alanine 3.76 4.91 L-Arginine 3.84 5.02L-Serine 3.69 4.82 Glicine 2.81 3.67 Proline 4.52 5.91 L-Aspartic Acid5.76 7.53 Taurine 0.16 0.21 L-Tyrosine 4.70 6.14 L-Carnitine 0.10 0.13Glyceryl Dibehenate 9.12 11.92 Sodium Alginate 0.02 0.03 ethylcellulose3.37 4.40 Total 76.51 100.00³ ¹corresponding to 4.84 g of L-Lysine base²corresponding to 2.49 g of L-Histidine base ³corresponding to 98.21 gif L-Lysine and L-Histidine are considered as base

Manufacturing Equipment Used for the Preparation of the Product

Equipment used for the production:

-   -   Balances of various types    -   ProCepT Mipro 900 ml set-up    -   Sieves    -   Static oven    -   ProCepT Fluid bed bottom-up coating (melt nozzle) (or any other        apparatus suitable for the granulate hot melt coating)

Amino Acids Mixture

Mix all amino acids in a suitable apparatus

Granulate Preparation

Transfer the amino acid mixture into the ProCepT Mipro, add thegranulating solution composed of a 2% sodium alginate water solution andgranulate. After the granulation sieve the granulate; use the fractionhaving 0.5 mm<PSD<1 mm for the next step.

Coating with Ethylcellulose (Polymer Coating)

Transfer the sieved fraction of granulate in the fluid bed apparatus andspray coat it with a methanol solution at 15% (w/w) of ethylcellulose.The total amount of polymer added should equal approximately 5.00% ofthe weight of the granulate.

Coating with Glyceryl Dibehenate (Hot Melt Coating)

Transfer the granulate coated with ethylcellulose in the fluid bedapparatus and spray coat it with melted glyceryl dibehenate. The totalamount of polymer added should equal approximately 15.00% of thegranulate weight.

Dissolution Test on the Product

-   Dissolution Medium: Medium pH 1.2±0.1 (0.1N Hydrochloric acid)-   Apparatus: Paddle Apparatus (Apparatus 2, USP <711> modified); 50    rpm-   Time: 30-60-120-180 minutes-   Temperature: 37±0.5° C.-   Volume Medium: 500 mL-   Sample: 2.0 g of Amino Acid mixture-   Ponderal dissolution test: 30-60-120-180 minutes

The results of the Ponderal Dissolution Test takes into account thatethyl cellulose and glyceryl dibehenate are not soluble in HCl solution.At 30-60-120-180 minutes the dissolution medium were filtered through apaper filter under vacuum. The filtered powder and filter were dried for±4 hours in a vacuum oven at 50° C. until constant weight achieved andthe samples were the weighed. For difference the released quantity ofamino acids was calculated and reported in FIG. 6.

Example 11—Manufacturing and Dissolution Testing of Free Amino AcidFormulation and Modified Release Formulations

Table 10a reports the qualitative and quantitative composition of arepresentative amino acid formulation AA mixture (bkT037/71).

TABLE 10a Amino Acid % (with HCl) Amino Acid % (As Base) L-Alanine 5.87L-Alanine 5.87 L-Arginine 6.01 L-Arginine 6.01 L-Cystine 1.99 L-Cystine1.99 L-Glutamine 12.22 L-Glutamine 12.22 Glicine 4.38 Glicine 4.38L-Histidine HCl 3.67 L-Histidine 2.98 L-Aspartic Acid 9.00 L-AsparticAcid 9.00 L-Proline 7.06 L-Proline 7.06 L-Serine 5.77 L-Serine 5.77Taurine 0.24 Taurine 0.24 L-Tyrosine 7.35 L-Tyrosine 7.35 L-Carnitine0.16 L-Carnitine 0.16 L-Isoleucine 5.00 L-Isoleucine 5.00 L-Lysine HCl7.23 L-Lysine 5.78 L-Treonine 5.17 L-Treonine 5.17 L-Tryptophan 1.64L-Tryptophan 1.64 L-Leucine 8.98 L-Leucine 8.98 L-Valine 6.25 L-Valine6.25 L-Methionine 2.00 L-Methionine 2.00 Total 100 Total 97.86

Manufacturing Method (General)

Manufacturing equipment used for the preparation of the Product

-   -   Balances of various types    -   Sieves    -   Static oven    -   Mixing machine        Sieve and mix all amino acids in a suitable apparatus        Amino Acids are mechanically passed through a 350 μm stainless        sieve and mixed for 30 minutes in the mixer.

Dissolution Test on the Product

-   Dissolution Medium: Medium pH 1.2±0.1 (0.1N Hydrochloric acid)-   Apparatus: Paddle Apparatus (Apparatus 2, USP <711> modified) 50 rpm-   Time: 30-60 minutes-   Temperature: 37±0.5° C.-   Volume Medium: 500 mL-   Sample: 2.0 g of Amino Acid mixture-   Ponderal dissolution test: 30-60-120-180 minutes    Each time point has its own dissolution vessel. At 30-60-120-180    minutes the dissolution medium were filtered through a paper filter    under vacuum. The filtered powder and filter were dried for ±4 hours    in a vacuum oven at 50° C. until constant weight achieved and the    samples were the weighed. For difference the released quantity of    amino acids was calculated.

Results

The results of ponderal dissolution testing are report in Table 10b andFIG. 7.

TABLE 10b Time (min) % AA released 0 0 30 99.96 60 100.00 120 99.95 18099.99

Analytical Conditions:

-   -   HPLC: Agilent series 1200    -   Detector: Fluorimeter Agilent series 1200    -   Chromatographic Column: YMC-Triart C18 12 nm S (250×4.6 mm—5 μm)        Amino Acids are analyzed after extraction (with different ways)        and derivatization with FMOC (9-Fluorenyl methyl chloroformate).        Note: Carnitina is analyzed by LC/MS.        The results of the dissolution testing for AA Mixture        (bkT037/71) are reported in Table 10c and FIG. 8.

TABLE 10c Time (min) Amino Acid 0 30 60 Alanine 0 111.8 109.0 Arginine 0110.1 107.0 Glutammine 0 85.9 84.3 Glycine 0 112.6 109.4 Hystidine 0102.7 96.3 Aspartic Acid 0 103.7 106.6 Proline 0 109.9 108.1 Serine 0108.1 105.2 Taurine 0 120.0 125.8 Tirosine 0 103.9 101.2 Carnitine 0122.5 121.3 Isoleucine 0 103.6 100.6 Lysine 0 103.8 101.6 Treonine 097.7 96.3 Triptophan 0 100.0 100.6 Leucine 0 110.1 106.6 Valine 0 105.4103.4 Methionine 0 107.0 104.5

Table 10d reports the quantitative and qualitative formulations of fourdifferent batches of ethylcellulose coated amino acid particles.

TABLE 10d APR Batch 1 APR Batch 2 APR Batch 3 APR Batch 4 (bkT037/(bkT037/ (bkT037/ (bkT037/ Component 72-1) 72-2) 72-3) 72-4) PSD 0.5 mm< 0.5 mm < 1.0 mm < 1.0 mm < fraction < fraction < fraction < fraction <1 mm 1 mm 1.6 mm 1.6 mm Alanine 5.57 5.43 5.57 5.43 Arginine 5.70 5.555.70 5.55 Cystine 1.89 1.84 1.89 1.84 Glutamine 11.60 11.30 11.60 11.30Glicine 4.16 4.05 4.16 4.05 Histidine HCl 3.48 3.39 3.48 3.39 AsparticAcid 8.55 8.32 8.55 8.32 Proline 6.70 6.52 6.70 6.52 Serine 5.48 5.345.48 5.34 Taurine 0.23 0.23 0.23 0.23 Tyrosine 6.98 6.79 6.98 6.79Carnitine 0.15 0.14 0.15 0.14 Isoleucine 4.75 4.62 4.75 4.62 Lysine HCl6.86 6.68 6.86 6.68 Threonine 4.90 4.78 4.90 4.78 Tryptophan 1.56 1.521.56 1.52 Leucine 8.53 8.30 8.53 8.30 Valine 5.94 5.78 5.94 5.78Methionine 1.90 1.85 1.90 1.85 Sodium 0.07 0.07 0.07 0.07 alginateEthylcellulose 5.00 7.50 5.00 7.50 Total 100.00 100.00 100.00 100.00

Manufacturing Method (General) Manufacturing Equipment Used for thePreparation of the Product

-   -   Balances of various types    -   ProCepT Mipro 900 ml set-up    -   Sieves    -   Static oven    -   ProCepT Fluid bed bottom-up coating (melt nozzle) (or any other        apparatus suitable for the granulate hot melt coating)        Amino Acids mixture

Mix all amino acids in a suitable apparatus.

Granulate Preparation

Transfer the amino acid mixture into the ProCepT Mipro. add thegranulating solution composed of a 2% sodium alginate water solution andgranulate. After the granulation sieve the granulate; use the fractionhaving 0.5 mm<PSD<1 mm and 0.5 mm<PSD<1.5 mm for the next step.

Coating with Ethylcellulose (Polymer Coating)

Transfer the sieved fraction of the granulate to the fluid bed apparatusand spray coat it with an ethanol solution at 15% (w/w) ofethylcellulose. The total amount of polymer added should equalapproximately 5.00% and 7.50% of the granulate weight.

Dissolution Test on the Product

Dissolution Conditions:

-   Dissolution Medium: Medium pH 1.2±0.1 (0.1N Hydrochloric acid)-   Apparatus: Paddle Apparatus (Apparatus 2. USP <711> modified); 50    rpm-   Time: 30-60-120-180-240 minutes-   Temperature: 37±0.5° C.-   Volume Medium: 500 mL-   Sample: 2.0 g of Amino Acid mixture-   Ponderal Dissolution test: Sampling Time: 30-60-120-180 minutes    -   Each time point has his own dissolution vessel.        At 30-60-120-180 minutes the dissolution medium were filtered        through a paper filter under vacuum. The filtered powder and        filter were dried for ±4 hours in a vacuum oven at 50° C. until        constant weight achieved and the samples were the weighed. For        difference the released quantity of amino acids was calculated.        Results

The results of ponderal dissolution testing are reported in Table 10eand FIG. 9.

TABLE 10e Batch Time (min) % Aas released APR Batch 1 bkT037/72-1) 0 030 59 60 73 120 86 180 90 APR Batch 2 (bkT037/72-2) 0 0 30 59 60 70 12082 180 90 APR Batch 3 (bkT037/72-3) 0 0 30 57 60 70 120 82 180 88 APRBatch 4 (bkT037/72-4) 0 0 30 44 60 59 120 62 180 81The analyses of the release of single amino acids have been performedonly on prototypes APR Batch 2 and APR Batch 4

Analytical Conditions:

-   -   HPLC: Agilent series 1200    -   Detector: Fluorimeter Agilent series 1200    -   Chromatographic Column: YMC-Triart C18 12 nm S (250×4.6 mm—5 μm)        Amino Acids are analyzed after extraction (with different ways)        and derivatization with FMOC (9-Fluorenyl methyl chloroformate).        Note: Carnitina is analyzed by LC/MS.

Results (Single Amino Acids)

The results of the dissolution testing for APR Batch 2 (bkT037/72-2) arereported in Table 10f and FIG. 10.

TABLE 10f Time (min) Amino Acid 0 30 60 120 180 240 Alanine 0 80.1 94.1100.0 102.0 102.9 Arginine 0 113.7 111.9 109.7 108.8 109.2 Glutammine 053.2 70.7 83.7 82.8 82.5 Glycine 0 85.9 94.3 95.6 96.3 96.3 Hystidine 084.0 102.2 104.0 108.4 105.5 Aspartic Acid 0 48.1 57.9 71.8 84.0 91.2Proline 0 104.1 105.8 102.3 101.8 101.5 Serine 0 91.2 97.6 98.7 98.398.7 Taurine 0 85.7 89.6 90.0 82.2 83.5 Tirosine 0 4.5 6.8 12.4 23.032.1 Carnitine 0 128.6 112.9 108.6 121.4 112.1 Isoleucine 0 28.8 48.972.9 85.9 91.8 Lysine 0 98.1 103.4 102.8 102.2 101.7 Treonine 0 77.493.5 102.7 102.7 104.6 Triptophan 0 82.2 90.8 92.8 94.1 92.8 Leucine 033.0 53.1 76.9 89.8 95.8 Valine 0 65.9 83.6 94.1 98.6 100.2 Methionine 069.7 77.8 87.6 94.1 97.8

The results of the dissolution testing for APR Batch 4 (bkT037/72-4) arereported in Table 10g and FIG. 11.

TABLE 10g Time (min) Amino Acid 0 30 60 120 180 240 Alanine 0 63.2 79.292.3 98.5 97.1 Arginine 0 89.9 98.7 103.4 106.5 107.7 Glutammine 0 35.648.8 67.1 76.5 79.5 Glycine 0 73.6 86.9 94.6 98.8 97.8 Hystidine 0 74.281.8 103.6 106.5 94.9 Aspartic Acid 0 42.3 54.7 68.9 80.2 86.1 Proline 092.5 100.6 105.4 108.0 106.7 Serine 0 78.1 91.4 95.9 100.4 99.1 Taurine0 60.0 68.7 74.3 83.9 82.2 Tirosine 0 3.2 6.5 5.8 9.4 11.1 Carnitine 0114.3 121.4 107.1 108.6 125.0 Isoleucine 0 15.6 28.8 47.0 63.4 73.2Lysine 0 88.8 97.8 101.5 104.3 103.6 Treonine 0 60.0 78.0 96.2 104.2105.2 Triptophan 0 65.7 77.6 86.2 91.4 91.4 Leucine 0 18.4 31.6 49.466.0 74.9 Valine 0 42.9 60.0 77.9 90.1 94.1 Methionine 0 43.2 61.6 67.688.1 85.4

Example 12—Manufacturing and Dissolution Testing of Modified ReleaseFormulations Using Ethylcellulose and Glyceryl Dibehenate as CoatingAgents

Table 11a reports the qualitative and quantitative composition of 4separate amino acid formulation coated with ethylcellulose and glyceryldibehenate as release rate modifying agents.

TABLE 11a APR Batch 5 APR Batch 6 APR Batch 7 APR Batch 8 (bkT037/(bkT037/ (bkT037/ (bkT037/ Component 73-5) 73-6) 73-7) 73-8) PSD 0.5 mm< 0.5 mm < 1.0 mm < 1.0 mm < fraction < fraction < fraction < fraction <1 mm 1 mm 1.6 mm 1.6 mm Alanine 5.00 4.87 5.00 4.87 Arginine 5.12 4.985.12 4.98 Cystine 1.69 1.65 1.69 1.65 Glutamine 10.41 10.13 10.41 10.13Glicine 3.73 3.63 3.73 3.63 Histidine HCl 3.12 3.04 3.12 3.04 AsparticAcid 7.67 7.46 7.67 7.46 Proline 6.01 5.85 6.01 5.85 Serine 4.92 4.794.92 4.79 Taurine 0.21 0.20 0.21 0.20 Tyrosine 6.26 6.09 6.26 6.09Carnitine 0.13 0.13 0.13 0.13 Isoleucine 4.26 4.15 4.26 4.15 Lysine HCl6.16 6.00 6.16 6.00 Threonine 4.40 4.28 4.40 4.28 Tryptophan 1.40 1.361.40 1.36 Leucine 7.65 7.45 7.65 7.45 Valine 5.32 5.18 5.32 5.18Methionine 1.70 1.65 1.70 1.65 Sodium 0.06 0.06 0.06 0.06 alginateEthylcellulose 4.49 6.71 4.49 6.71 Glyceryl 10.0 10.00 10.00 10.00dibehenate Total 100.00 100.00 100.00 100.00

Manufacturing Method (General) Manufacturing Equipment Used for thePreparation of the Product

-   -   Balances of various types    -   ProCepT Mipro 900 ml set-up    -   Sieves    -   Static oven    -   ProCepT Fluid bed bottom-up coating (melt nozzle) (or any other        apparatus suitable for the granulate hot melt coating)

Amino Acid Mixture

Mix all amino acids in a suitable apparatus

Granulate Preparation

Transfer the amino acids mixture into the ProCepT Mipro. add thegranulating solution composed of a 2% sodium alginate water solution andgranulate. After the granulation sieve the granulate; use the fractionhaving 0.5 mm<PSD<1 mm or 0.5 mm<PSD<1.6 mm for the next step.

Coating with Ethylcellulose (Polymer Coating)

Transfer the sieved fraction of granulate in the fluid bed apparatus andspray coat it with a methanol solution at 15% (w/w) of ethylcellulose.The total amount of polymer added should equal approximately 5.00% and7.50% of the granulate weight.

Coating with Glyceryl Dibehenate (Hot Melt Coating)

Transfer the granulate coated with ethylcellulose in the fluid bedapparatus and spray coat it with melted glyceryl dibehenate. The totalamount of polymer added should equal approximately 10.00% of thegranulate weight.

Analytical Conditions:

Dissolution Test on the Product

-   Dissolution Medium: Medium pH 1.2±0.1 (0.1N Hydrochloric acid)-   Apparatus: Paddle Apparatus (Apparatus 2. USP <711> modified); 50    rpm-   Time: 30-60-120-180-240 minutes-   Temperature: 37±0.5° C.-   Volume Medium: 500 mL-   Sample: 2.0 g of Amino Acid mixture-   Ponderal dissolution test: Sampling Time: 30-60-120-180 minutes At    30-60-120-180 minutes the dissolution medium were filtered through a    paper filter under vacuum. The filtered powder and filter were dried    for ±4 hours in a vacuum oven at 50° C. until constant weight    achieved and the samples were the weighed. For difference the    released quantity of amino acids was calculated

Results

The results of the ponderal dissolution testing are reported in Table11b and FIG. 12.

TABLE 11b Batch Time (min) % Aas released APR Batch 5 (bkT037/73-5) 0 030 40 60 53 120 67 180 77 APR Batch 6 (bkT037/73-6) 0 0 30 38 60 50 12062 180 69 APR Batch 7 (bkT037/73-7) 0 0 30 26 60 37 120 55 180 63 APRBatch 8 (bkT037/73-8) 0 0 30 21 60 29 120 47 180 56The analyses have been performed on prototypes APR Batch 5 and APR Batch7

Analytical Conditions:

-   -   HPLC: Agilent series 1200    -   Detector: Fluorimeter Agilent series 1200    -   Chromatographic Column: YMC-Triart C18 12 nm S (250×4.6 mm—5 μm)        Amino Acids are analyzed after derivatization with FMOC        (9-Fluorenyl methyl chloroformate)        Note: Carnitina is analyzed by LC/MS.

Results (Single Amino Acids)

The single amino acid dissolution test results for APR Batch 5(bkT037/73-5) are reported in Table 11c and FIG. 13.

TABLE 11c Time (min) Amino Acid 0 30 60 120 180 240 Alanine 0 54.4 82.0102.2 102.0 102.2 Arginine 0 84.6 97.1 106.1 104.9 104.1 Glutammine 038.5 52.6 84.9 92.9 93.9 Glycine 0 64.9 84.7 96.8 95.7 102.4 Hystidine 039.3 79.1 79.5 81.1 83.1 Aspartic Acid 0 34.8 48.2 65.1 76.0 83.4Proline 0 87.7 97.2 101.8 99.2 98.7 Serine 0 70.1 86.6 96.1 95.5 95.1Taurine 0 81.4 119.5 112.4 126.2 119.0 Tirosine 0 2.4 6.9 12.0 21.2 26.4Carnitine 0 88.5 100.0 109.2 111.5 110.8 Isoleucine 0 11.2 26.8 58.775.8 82.6 Lysine 0 85.2 93.9 100.2 98.2 98.4 Treonine 0 47.5 74.1 95.597.3 100.2 Triptophan 0 68.8 77.9 87.9 96.4 90.7 Leucine 0 13.9 30.560.8 79.1 85.6 Valine 0 35.0 58.8 86.3 91.7 94.5 Methionine 0 47.4 73.587.6 91.2 99.4

The single amino acid dissolution test results for APR Batch 7(bkT037/72-7) are reported in Table 11d and FIG. 14.

TABLE 11d Time (min) Amino Acid 0 30 60 120 180 240 Alanine 0 39.0 60.886.8 95.0 96.0 Arginine 0 60.2 74.8 92.6 106.1 98.8 Glutammine 0 27.037.4 69.5 81.9 84.1 Glycine 0 47.5 66.0 88.2 96.5 94.6 Hystidine 0 44.558.3 79.1 79.1 75.6 Aspartic Acid 0 27.4 37.2 57.0 70.3 75.9 Proline 067.1 79.0 95.8 91.2 98.7 Serine 0 51.4 67.7 87.2 94.3 93.3 Taurine 062.4 81.0 114.3 109.5 110.5 Tirosine 0 2.4 4.9 8.9 19.3 20.9 Carnitine 068.0 82.3 96.9 98.5 96.9 Isoleucine 0 5.8 15.0 39.7 61.3 69.7 Lysine 062.6 76.6 93.1 94.9 96.3 Treonine 0 31.6 54.3 84.1 94.8 98.2 Triptophan0 45.7 63.2 82.1 86.4 90.7 Leucine 0 7.1 17.5 41.7 62.5 71.2 Valine 019.0 36.5 66.9 83.3 86.1 Methionine 0 31.9 48.0 77.6 75.9 88.2

Table 11e and FIG. 15 report the total amino acids released (PonderalDissolution Test) at various time points from each of the four testformulations. compared to an immediate release amino acid formulation.

TABLE 11e Batch Time (min) % Aas released APR Batch 2 (bkT037/72-2) 0 030 59 60 70 120 82 180 90 APR Batch 4 (bkT037/72-4) 0 0 30 44 60 59 12062 180 81 APR Batch 5 (bkT037/73-5) 0 0 30 40 60 53 120 67 180 77 APRBatch 7 (bkT037/73-7) 0 0 30 26 60 37 120 55 180 63 AA mixture(bkT037/71) 0 0 30 100 60 100 120 100 180 100

Tables 11f-11j report the quantity of individual amino acids releasedfrom the four different test formulation at distinct time points.

TABLE 11f 30 Minutes APR APR APR APR Free AA Batch 2 Batch 4 Batch 5Batch 7 (bkT037/ (bkT037/ (bkT037/ (bkT037/ (bkT037/ Amino Acid 71)72-2) 72-4) 73-5) 73-7) Alanine 111.8 80.1 63.2 54.4 39.0 Arginine 110.1113.7 89.9 84.6 60.2 Glutammine 85.9 53.2 35.6 38.5 27.0 Glycine 112.685.9 73.6 64.9 47.5 Hystidine 102.7 84.0 74.2 39.3 44.5 Aspartic 103.748.1 42.3 34.8 27.4 Acid Proline 109.9 104.1 92.5 87.7 67.1 Serine 108.191.2 78.1 70.1 51.4 Taurine 120.0 85.7 60.0 81.4 62.4 Tirosine 103.9 4.53.2 2.4 2.4 Carnitine 122.5 128.6 114.3 88.5 68.0 Isoleucine 103.6 28.815.6 11.2 5.8 Lysine 103.8 98.1 88.8 85.2 62.6 Treonine 97.7 77.4 60.047.5 31.6 Triptophan 100.0 82.2 65.7 38.8 45.7 Leucine 110.1 33.0 18.413.9 7.1 Valine 105.4 65.9 42.9 35.0 19.0 Methionine 107.0 69.7 43.247.4 31.9

TABLE 11g 60 Minutes APR APR APR APR Free AA Batch 2 Batch 4 Batch 5Batch 7 (bkT037/ (bkT037/ (bkT037/ (bkT037/ (bkT037/ Amino Acid 71)72-2) 72-4) 73-5) 73-7) Alanine 109.0 94.1 79.2 82.0 60.8 Arginine 107.0111.9 98.7 97.1 74.8 Glutammine 84.3 70.7 48.8 52.6 37.4 Glycine 109.494.3 86.9 84.7 66.0 Hystidine 96.3 102.2 81.8 79.1 58.3 Aspartic 106.657.9 54.7 48.2 37.2 Acid Proline 108.1 105.8 100.6 97.2 79.0 Serine105.2 97.6 91.4 86.6 67.7 Taurine 125.8 89.6 68.7 119.5 81.0 Tirosine101.2 6.8 6.5 6.9 4.9 Carnitine 121.3 112.9 121.4 100.0 82.3 Isoleucine100.6 48.9 28.8 26.8 15.0 Lysine 101.6 103.4 97.8 93.9 76.6 Treonine96.3 93.5 78.0 74.1 54.3 Triptophan 100.6 90.8 77.6 77.9 63.2 Leucine106.6 53.1 31.6 30.5 17.5 Valine 103.4 83.6 60.0 58.8 36.5 Methionine104.5 77.8 61.6 73.5 48.0

TABLE 11h 120 Minutes APR Batch 2 APR Batch 4 APR Batch 5 APR Batch 7(bkT037/ (bkT037/ (bkT037/ (bkT037/ Amino Acid 72-2) 72-4) 73-5) 73-7)Alanine 100.0 92.3 102.2 86.8 Arginine 109.7 103.4 106.1 92.6 Glutammine83.7 67.1 84.9 69.5 Glycine 95.6 94.6 96.8 88.2 Hystidine 104.0 103.679.5 79.1 Aspartic Acid 71.8 68.9 65.1 57.0 Proline 102.3 105.4 101.895.8 Serine 98.7 95.9 96.1 87.2 Taurine 90.0 74.3 112.4 114.3 Tirosine12.4 5.8 12.0 8.9 Carnitine 108.6 107.1 109.2 96.9 Isoleucine 72.9 47.058.7 39.7 Lysine 102.8 101.5 100.2 93.1 Treonine 102.7 96.2 95.5 84.1Triptophan 92.8 86.2 87.9 82.1 Leucine 76.9 49.4 60.8 41.7 Valine 94.177.9 86.3 66.9 Methionine 87.6 67.6 87.6 77.6

TABLE 11i 180 Minutes APR Batch 2 APR Batch 4 APR Batch 5 APR Batch 7(bkT037/ (bkT037/ (bkT037/ (bkT037/ 72-2) 72-4) 73-5) 73-7) Alanine102.0 98.5 102.0 95.0 Arginine 108.8 106.5 104.9 106.1 Glutammine 82.876.5 92.9 81.9 Glycine 96.3 98.8 95.7 96.5 Hystidine 108.4 106.5 81.179.1 Aspartic Acid 84.0 80.2 76.0 70.3 Proline 101.8 108.0 99.2 91.2Serine 98.3 100.4 95.5 94.3 Taurine 82.2 83.9 126.2 109.5 Tirosine 23.09.4 21.2 19.3 Carnitine 121.4 108.6 111.5 98.5 Isoleucine 85.9 63.4 75.861.3 Lysine 102.2 104.3 98.2 94.9 Treonine 102.7 104.2 97.3 94.8Triptophan 94.1 91.4 96.4 86.4 Leucine 89.8 66.0 79.1 62.5 Valine 98.690.1 91.7 83.3 Methionine 94.1 88.1 91.2 75.9

TABLE 11j 240 Minutes APR Batch 2 APR Batch 4 APR Batch 5 APR Batch 7(bkT037/ (bkT037/ (bkT037/ (bkT037/ 72-2) 72-4) 73-5) 73-7) Alanine102.9 97.1 102.2 96.0 Arginine 109.2 107.7 104.1 98.8 Glutammine 82.579.5 93.9 84.1 Glycine 96.3 97.8 102.4 94.6 Hystidine 105.5 94.9 83.175.6 Aspartic Acid 91.2 86.1 83.4 75.9 Proline 101.5 106.7 98.7 98.7Serine 98.7 99.1 95.1 93.3 Taurine 83.5 82.2 119.0 110.5 Tirosine 32.111.1 26.4 20.9 Carnitine 112.1 125.0 110.8 96.9 Isoleucine 91.8 73.282.6 69.7 Lysine 101.7 103.6 98.4 96.3 Treonine 104.6 105.2 100.2 98.2Triptophan 92.8 91.4 90.7 90.7 Leucine 95.8 74.9 85.6 71.2 Valine 100.294.1 94.5 86.1 Methionine 97.8 85.4 99.4 88.2

Example 13—Final Amino Acids Composition

The quantitative composition for phenylketonuria of the amino acid mixhas been optimized according to the nutritionists' recommendations; thepercentages of the single amino acid are reported in Table 12a.

TABLE 12a Amino Acid % (with HCl) Amino Acid % (As Base) L-Alanine 2.9L-Alanine 2.9 L-Arginine 3.9 L-Arginine 3.9 L-Cystine 2.0 L-Cystine 2.0L-Glutamine 19.5 L-Glutamine 19.5 Glicine 4.9 Glicine 4.9 L-HistidineHCl 3.4 L-Histidine 2.76 L-Aspartic Acid 5.9 L-Aspartic Acid 5.9L-Proline 5.9 L-Proline 5.9 L-Serine 3.3 L-Serine 3.3 Taurine 0.3Taurine 0.3 L-Tyrosine 9.8 L-Tyrosine 9.8 L-Carnitine 0.1 L-Carnitine0.1 L-Isoleucine 5.4 L-Isoleucine 5.4 L-Lysine HCl 8.5 L-Lysine 6.80L-Treonine 4.9 L-Treonine 4.9 L-Tryptophan 2.0 L-Tryptophan 2.0L-Leucine 11.2 L-Leucine 11.2 L-Valine 4.9 L-Valine 4.9 L-Methionine 1.4L-Methionine 1.4 Total 100 Total 98

Example 14—In Vivo Bioavailability in Pig

An in vivo bioavailability study was undertaken in pigs in order tomodel the anticipated pharmacokinetics and metabolism of theformulations of the present invention, as compared to a comparablemixture of free amino acids, having similar proportions and totalamounts of amino acids, and a commercially available formulation of milkproteins (casein). The following formulations were tested:

-   -   APR-04 (bkT037/72-4)    -   APR-07 (bkT037/73-7)    -   Free amino acid (bkT037/71)    -   Casein

In Vivo Study Design:

-   -   Subjects: 8 pigs    -   Design: 4 formulations in crossover    -   Administration route and method: oral, by gavage. The product        dose was administered to each animal mixed with a small amount        of water (300 ml) in order to ensure fast (≤5 minutes)        assumption. The total amount of the product was administered        using syringes. The animals were fasted for 13.5±0.5 hours        before the treatment and the access to water was suspended one        hour before and one hour after the treatment.    -   Dose, frequency and duration of administration: The amount of        product administered was based on body-weight and equaled 0.8 g        of amino acid/kg body weight for each test product. The products        were administered by a single administration in the morning of        each day of treatment.    -   Wash out: 48-72 hours    -   Blood sampling: 0.75 h before Treatment (T), 0.5 h before T,        0.25 h before T, 0.25 h after

T, 0.5 h after T, 0.75 h after T, 1 h after T, 1.25 h after T, 1.5 hafter T, 2 h after T, 2.5 h after T, 3 h after T, 4 h after T, 5 h afterT

-   -   Analysis: The plasma concentrations of 14 amino acids (Alanine,        Arginine, Glutamine,

Glycine, Histidine, Isoleucine, Leucine, Lysine, Proline, Serine,Threonine, Tryptophan, Tyrosine, Valine) were measured and analyzed.Methionine and Aspartic acid were excluded from the analysis due tostability issues. The following parameters were evaluated: Area Underthe concentration/time Curve (AUC0-last), peak concentration (C_(max)),time to peak concentration (T_(max)) and C_(last). The behavior of thetwo APR Formulations was compared to the formulation of Free Amino Acidsand Casein.

Results:

The plasma concentration of an amino acid (AA) is the result of itsrates of appearance (Ra) in and disappearance (Rd) from plasma. Factorscontrolling Ra include protein intake and tissue release. Factorscontrolling Rd include tissue uptake and body loss through urine, sweat,etc. Hormones also help to regulate plasma AA concentrations,particularly insulin and glucagon, both of which inducehypoaminoacidemia (but for quite different reasons), and cortisol, whichinduces hyperaminoacidemia. In addition, in pathologic states, catecholamines, thyroid hormones, and cytokines can modulate plasma AA levels.Peripheral availability of Aas after protein ingestion is controlled bythe liver, with an activation of ureagenesis in hyperprotein feeding andrepression during a hypoprotein diet (Cynober 2002). All these factorscan influence the plasma amino acid concentration reported herein.

1) Mean Aggregate Plasma Amino Acid Concentrations

The mean plasma concentrations of the 14 amino acids over time, on anaggregate basis, is reported in FIG. 16 and Table 14a. Basal values werenot subtracted prior to performing the calculations.

TABLE 14a 14 AA Free amino (RAW DATA) APR-04 APR-07 acids Casein AUC0-last 1184.1 1141.8 1200.7 1125.6 C_(max) 339.0 341.9 419.2 324.8C_(last) 190.8 168.7 163.5 172.9The following observations can be made:

-   -   The APR Formulations show a lower plasma peak of aggregate amino        acids (C_(max)) when compared to the free amino acid        formulation.    -   If we consider the curve of the single amino acids, Repeated        Measures ANOVA applied to Ln-transformed data show a significant        difference (p<0.05) in C_(max) from APR-04 versus Free Amino        Acids for Arginine, Isoleucine, Leucine, Tyrosine and Valine.    -   If we consider the curve of the single amino acids, Repeated        Measures ANOVA applied to Ln-transformed data show a significant        difference (p<0.05) in C_(max) from APR-07 versus Free Amino        Acids for Isoleucine, Leucine, Tyrosine and Valine.

The difference in C_(max) is even more evident is we consider the BCdata, in which the basal values of amino acids are subtracted, as shownbelow in Table 14b, although the difference is not statisticallysignificant for the mean of the entire aggregate of amino acids:

TABLE 14b 14 AA Free amino (BC DATA) APR-04 APR-07 acids Casein AUC0-last 332.3 302.3 382.9 258.9 C_(max) 168.6 172.5 251.5 147.3

-   -   The higher initial peak plasma concentration of amino acids        (C_(max)) observed for the Free Amino Acid Formulation rapidly        decreases, as evidenced by the “C_(last)” parameter. “C_(last)”        is the amino acid plasma concentration at the last sampling (5        h)    -   If we compare the curve of the single amino acids, in the group        treated with APR-04, the C_(last) is higher (but not        statistically significant except for Valine) versus the free        Amino Acid Group for Alanine, Arginine, Glutamine, Glycine,        Histidine, Isoleucine, Leucine, Lysine, Proline, Serine,        Threonine, Tryptophan and Valine (13 amino acids out of 14)    -   If we compare the curve of the single amino acids, in the group        treated with APR-07, the C_(last) is higher (but not        statistically significant except for Valine, Isoleucine and        Leucine) versus the free Amino Acid Group for Arginine,        Histidine, Isoleucine, Leucine, Lysine, Proline, Tryptophan, and        Valine (8 amino acids out of 14)    -   The trend in C_(max) and C_(last) observed in the plasma        concentration curve for aggregated amino acids becomes        statistically significant if the subgroup of Essential Amino        Acids and the subgroup of BCAAs (Branched Chain Amino Acids) is        considered (see points 2 and 3).

2) Mean of Analyzed Essential Amino Acid Concentrations (Arginine,Histidine, Isoleucine, Leucine, Threonine, Lysine, Tryptophan andValine).

The mean plasma concentrations of the essential amino acids tested, onan aggregate basis, is reported in FIG. 17 and Table 14c. The aminoacids aggregated together for analysis were Arginine, Histidine,Isoleucine, Leucine, Threonine, Lysine, Tryptophan and Valine. Thissubgroup was chosen for analysis because these amino acids cannot beendogenously synthesized. As a result, their concentrations in plasmafrom sampling the jugular vein may be more representative of theintestinal adsorption of the tested formulations than the non-essentialamino acids whose presence in blood could be independently generated bythe organism.

TABLE 14c ESSENTIAL AA Free amino (RAW DATA) APR-04 APR-07 acids CaseinAUC 0-last 1020.5 1029.0 1098.5 969.9 C_(max) 277.0 292.9 409.0 299.1T_(max) 1.50 1.19 0.88 1.16 C_(last) 170.7 166.2 138.2 153.0The statistical analysis for the date reported in Table 14c (for AUC,C_(max), and C_(last): one-way repeated measures ANOVA with post hocanalysis with Bonferroni adjustment; for T_(max): Friedman test followedby multiple sign test for pairwise comparison) is reported in Table 14d.

TABLE 14d RAW DATA AUC C_(max) T_(max) C_(last) Free AA vs APR-04 NS<0.01 NS 0.029 Free AA vs APR-07 NS <0.01 NS 0.017

3) Mean of BCAAs Concentration (Branched Chain Amino Acids: Valine,Isoleucine and Leucine).

The mean plasma concentrations of the branched chain amino acids tested,on an aggregate basis, is reported in FIG. 18 and Table 14e. The aminoacids aggregated together for analysis were valine, isoleucine andleucine. This subgroup of essential amino acids was chosen for analysisbecause these essential amino acids are unique among amino acids in thattheir first catabolic step cannot occur in the liver. As a consequence,they largely escape first-pass splanchnic metabolism (Brosnan et al).

TABLE 14e BCAA Free amino (RAW DATA) APR-04 APR-07 acids Casein AUC0-last 1365.8 1367.3 1556.1 1365.3 C_(max) 350.9 335.8 628.3 401.1T_(max) 2.41 1.72 0.63 1.16 C_(last) 253.7 256.2 197.0 228.7The statistical analysis for the date reported in Table 14e (for AUC,C_(max), and C_(last): one-way repeated measures ANOVA with post hocanalysis with Bonferroni adjustment; for T_(max): Friedman test followedby multiple sign test for pairwise comparison) is reported in Table 14f.

TABLE 14f RAW DATA AUC C_(max) T_(max) C_(last) Free AA vs APR-04 NS<0.01 <0.01 0.045 Free AA vs APR-07 NS <0.01 0.031 <0.01

4) Mean in Total LNAAs Concentration (Large Neutral Amino Acids:Phenylalanine, Tyrosine, Tryptophan, Threonine, Methionine, Valine,Isoleucine, Leucine, Histidine).

The plasma concentrations of the large neutral amino acids tested, on anaggregate basis, are reported in FIG. 19 and Table 14g. The amino acidsaggregated together for analysis were tryptophan, threonine, valine,isoleucine, leucine, histidine. Tyrosine was excluded because of theanomalous behavior it exhibited in the dissolution and in vivo testing,and methionine because it was not stable in the samples. Phenylalaninewas excluded from the analysis because it was not present in theformulations tested. This subgroup of essential amino acids was chosenfor analysis because LNAA share with phenylalanine a common transportsystem in order to enter the branched. High plasma concentration ofLNAAs may reduce the uptake into the branched (Van Spronsen et al).might thus bring clinical advantages. In particular, if the analysis onthe subgroup show an increased C_(last) for the APR formulations versusthe free amino acid formulations, it can be speculated that the patientmay have a sort of “prolonged protection/tolerance” to phenylalanine.

TABLE 14g LNAA Free amino (RAW DATA) APR-04 APR-07 acids Casein AUC0-last 925.0 921.8 1028.3 868.9 C_(max) 244.3 240.0 383.9 256.6 T_(max)2.16 1.22 0.69 1.16 C_(last) 166.9 161.8 132.5 143.0The statistical analysis for the date reported in Table 14g (for AUC,C_(max), and C_(last): one-way repeated measures ANOVA with post hocanalysis with Bonferroni adjustment; for T_(max): Friedman test followedby multiple sign test for pairwise comparison) is reported in Table 14h.

TABLE 14h RAW DATA AUC C_(max) T_(max) C_(last) Free AA NS <0.01 <0.010.031 vs APR-04 Free AA NS <0.01 NS (0.070) <0.01 vs APR-07

Throughout this application. various publications are referenced. Thedisclosures of these publications are hereby incorporated by referencein order to more fully describe the state of the art to which thisinvention pertains. It will be apparent to those skilled in the art thatvarious modifications and variations can be made in the presentinvention without departing from the scope or spirit of the invention.Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only. with a true scope and spiritof the invention being indicated by the following claims.

CITED REFERENCES

-   AMIDON G. L. LENNERNÄS H. SHAH V. P. CRISON J. R. A theoretical    basis for a biopharmaceutical drug classification: the correlation    of in vitro drug product dissolution and in vivo bioavailability.    Pharm. Res. 12:414-420 (1995)-   BARACOS V. E. Animal models of amino acids metabolism: a focus on    the intestine. J. Nutria. 134:1656S-1659S (2004)-   BROSNAN J T. BROSNAN M E. Branched-chain amino acids: enzyme and    substrate regulation. J Nutr. 2006 January; 136(1 Suppl):207S-11S.-   CYNOBER L A. Plasma amino acid levels with a note on membrane    transport: characteristics. regulation, and metabolic significance.    Nutrition. 2002 September; 18(9):761-6.-   DIOGUARDI F. S. Clinical use of amino acids as dietary supplement:    pros and cons. J. Cochexia Sarcopenia Muscle. 2:75-80 (2011)-   EMA 2010. Guideline on the investigation of bioequivalence    CPMP/EWP/QWP/1401/98 Rev. 1. London. 20 Jan. 2010-   GIOVANNINI M. RIVA E. SALVATICI E. CEFALO G. RADAELLI G. Randomized    controlled trial of a protein substitute with prolonged release on    the protein statutes of children with phenylketonuria. J. Am. Coll.    Nutr. 33. 103-110 (2014)-   GROPPER S. S. ACOSTA P. B. Effect of simultaneous ingestion of    L-amino acids and whole protein on plasma amino acid and urea    nitrogen concentrations in humans. J. Parenteral Enteral Nutr.    15:48-53 (1991)-   KEOHANE P. P. GRIMBLE G. K. BROWN B. SPILLER R. C. Influence of    protein composition and hydrolysis method on intestinal absorption    of protein in man. Gut. 26:907-913 (1985)-   MONTGOMERY R. DRYER R. L. CONWAY T. W. SPECTOR A. A. Biochimica.    Aspetti medico biologici. Translation by Berra B. Ragnotti G.    Tettamanti G. Edi-Ermes. Milan. 1988    -   Chapter 1: Nutrizione. pp. 1-47    -   Chapter 2: Struttura delle proteine. pp. 49-121    -   Chapter 10: Metabolismo degli amminoacidi. pp. 584-639.-   NEY D. M. BLANK R. D. HANSEN K. E. Advances in the nutritional and    pharmacological management of phenylketonuria. Co-Clinical    Nutrition. Com. 17:61-68 (2014)-   NEY DM. Does the PKU diet contribute to impaired renal function? J    Inherit Metab Dis. 2013 September; 36(5):903-4.-   PENA M J. ROCHA J C AND BORGES N. Amino Acids. Glucose Metabolism    and Clinical Relevance for Phenylketonuria Management. Ann Nutr    Disord & Ther—Volume 2 Issue 3-2015-   VAN SPRONSEN F J. DE GROOT M J. HOEKSMA M. REIJNGOUD D J. VAN    RIJN M. Large neutral amino acids in the treatment of PKU: from    theory to practice. J Inherit Metab Dis. 2010 December; 33(6):671-6.-   VLIET D. VAN. DERKS T. G. J. RIJN M. VAN. DE GROOT M. J. MCDONALD A.    et al. Single amino acid supplementation in aminoacidopatients: a    systemic review. Orphanet J. Rare Dis. 9:1-14 (2014)-   WAISBREN S. E. NOEL K. FAHRDACH K. CELLA C. FRAME D. DORENBAUM A.    LEVY H. Phenylalanine blood levels and clinical outcomes in    phenylketonuria: a systemic literature review and meta-analysis.    Mol. Genet. Metab. 92:63-70 (2007)-   Branched Chain Amino Acids in Clinical Nutrition—Volume 1. Raj    endram. R; Preedy. V. R.; Patel. V. B. (Eds) 2015. XXViii. 270 p. 91    illus. in color. Hardcover-   WHANG K. Y. AND EASTER R. A. Asian-Aus. J. Anim. Sci. 2000 Vol. 13.    No 6:811-816

1. An orally administered amino acid formulation comprising one or moremodified release amino acids wherein the formulation comprising 2 g ofthe modified release amino acids releases no more than x % of themodified release amino acids in 30 minutes of dissolution testingperformed in a <711> USP 39 NF 34, paddle apparatus, at 37° C., in 450or 500 mL, 0.1 N hydrochloric acid (pH 1.2), paddle speed 50 rpm,wherein x % is 90%, 80%, 70%, 60%, 50% or even 40%.
 2. An orallyadministered amino acid formulation comprising one or more modifiedrelease amino acids selected from isoleucine, leucine, tyrosine, valine,and combinations thereof, wherein the formulation comprising 2 g of themodified release amino acids releases no more than x % of the modifiedrelease amino acids in 30 minutes of dissolution testing performed in a<711> USP 39 NF 34, paddle apparatus, at 37° C., in 450 or 500 mL, 0.1 Nhydrochloric acid (pH 1.2), paddle speed 50 rpm, wherein x % is 90%,80%, 70%, 60%, 50%, or even 40%.
 3. An orally administered amino acidformulation comprising one or more modified release brain chain aminoacids, wherein the formulation comprising 2 g of the modified releaseamino acids releases no more than x % of the modified release aminoacids in 30 minutes of dissolution testing performed in a <711> USP 39NF 34, paddle apparatus, at 37° C., in 450 or 500 mL, 0.1 N hydrochloricacid (pH 1.2), paddle speed 50 rpm, wherein x % is 90%, 80%, 70%, 60%,50% or even 40%.
 4. The formulation of claim 3, wherein the formulationcomprises modified release valine, isoleucine and leucine
 5. An orallyadministered amino acid formulation comprising a combination of modifiedrelease essential amino acids wherein the formulation comprising 2 g ofthe modified release amino acids releases no more than x % of themodified release amino acids in 30 minutes of dissolution testingperformed in a <711> USP 39 NF 34, paddle apparatus, at 37° C., in 450or 500 mL, 0.1 N hydrochloric acid (pH 1.2), paddle speed 50 rpm,wherein x % is 90%, 80%, 70%, 60%, 50% or even 40%.
 6. The formulationof claim 5, wherein the formulation comprises modified release Arginine,Histidine, Isoleucine, Leucine, Threonine, Lysine and Tryptophan.
 7. Anorally administered amino acid formulation comprising modified releaselarge neutral amino acids, wherein the formulation comprising 2 g of themodified release large neutral amino acids releases no more than x % ofthe modified release amino acids in 30 minutes of dissolution testingperformed in a <711> USP 39 NF 34, paddle apparatus, at 37° C., in 450or 500 mL, 0.1 N hydrochloric acid (pH 1.2), paddle speed 50 rpm,wherein x % is 90%, 80%, 70%, 60%, 50% or even 40%.
 8. The formulationof claim 7, wherein the formulation comprises modified releasetryptophan, threonine, valine, isoleucine, leucine, and histidine.
 9. Anorally administered amino acid formulation comprising a combination ofmodified release non-essential amino acids wherein the formulationcomprising 2 g of the modified release amino acids releases no more thanx % of the modified release amino acids in 30 minutes of dissolutiontesting performed in a <711> USP 39 NF 34, paddle apparatus, at 37° C.,in 450 or 500 mL, 0.1 N hydrochloric acid (pH 1.2), paddle speed 50 rpm,wherein x % is 90%, 80%, 70%, 60%, 50% or even 40%.
 10. The formulationof claim 1, comprising five or more of the following amino acids asmodified release amino acids in the recited weight parts: Amino AcidWeight Parts L-Alanine 2.0-7.0 L-Arginine 3.0-8.2 L-Aspartic Acid 5.0-10.5 L-Cystine 1.5-4.0 L-Glutamic Acid + 10.0-22.0 GlutamineGlycine 4.0-8.0 L-Histidine 2.5-5.0 L-Isoleucine 4.0-7.0 L-Leucine 8.0-14.0 L-Lysine 4.9-9.0 L-Methionine 1.0-3.0 L-Phenylalanine 4.5-7.0L-Proline  4.0-10.0 L-Serine 3.0-7.0 L-Threonine 4.0-6.5 L-Tryptophan1.5-3.0 L-Tyrosine  6.5-12.0 L-Valine 4.0-8.0 L-Carnitine 0.05-0.2 Taurine 0.1-0.7


11. The formulation of claim 10, comprising ten or more of the recitedamino acids as modified release amino acids in the recited weight parts.12. The formulation of claim 10, comprising fifteen or more of therecited amino acids as modified release amino acids in the recitedweight parts.
 13. The formulation of claim 10, comprising all of therecited amino acids in the recited weight parts.
 14. The formulation ofclaim 10, comprising the recited large neutral amino acids as modifiedrelease amino acids in the recited weight parts.
 15. The formulation ofclaim 10, comprising the recited essential amino acids as modifiedrelease amino acids in the recited weight parts.
 16. The formulation ofclaim 10, comprising the recited brain chain amino acids as modifiedrelease amino acids in the recited weight parts.
 17. The formulation ofclaim 1, comprising five or more of the following amino acids asmodified release amino acids in the recited weight parts: Amino AcidWeight Parts L-Alanine 2.0-7.0 L-Arginine 3.0-8.0 L-Aspartic Acid 5.0-10.5 L-Cystine 1.0-3.0 L-Glutamic Acid + 11.0-22.0 GlutamineGlycine 4.0-6.0 L-Histidine 2.0-3.5 L-Isoleucine 4.5-6.5 L-Leucine 8.0-13.0 L-Lysine 4.9-8.0 L-Methionine 1.0-3.0 L-Phenylalanine 0.0L-Proline 5.0-8.2 L-Serine 3.0-7.0 L-Threonine 4.0-6.0 L-Tryptophan1.5-3.0 L-Tyrosine  6.5-12.0 L-Valine 4.0-8.0 L-Carnitine 0.05-0.2 Taurine 0.2-0.5


18. The formulation of claim 17, comprising ten or more of the recitedamino acids as modified release amino acids in the recited weight parts.19. The formulation of claim 17, comprising fifteen or more of therecited amino acids as modified release amino acids in the recitedweight parts.
 20. The formulation of claim 17, comprising all of therecited amino acids in the recited weight parts.
 21. The formulation ofclaim 17, comprising the recited large neutral amino acids as modifiedrelease amino acids in the recited weight parts.
 22. The formulation ofclaim 17, comprising the recited essential amino acids as modifiedrelease amino acids in the recited weight parts.
 23. The formulation ofclaim 17, comprising the recited brain chain amino acids as modifiedrelease amino acids in the recited weight parts.
 24. The formulation ofclaim 1, comprising five or more of the following amino acids asmodified release amino acids in the recited weight parts Amino AcidWeight Parts L-Alanine 3.5-5.0 L-Arginine 7.0-8.0 L-Aspartic Acid6.0-8.0 L-Cystine 2.5-3.5 L-Glutamic Acid +  8.0-12.0 Glutamine Glycine6.0-8.0 L-Histidine 4.0-5.0 L-Isoleucine 6.0-8.0 L-Leucine 10.0-12.5L-Lysine 7.5-9.5 L-Methionine 1.5-2.5 L-Phenylalanine 0.0 L-Proline7.5-9.0 L-Serine 4.5-6.0 L-Threonine 5.0-6.5 L-Tryptophan 1.5-3.0L-Tyrosine 0.0 L-Valine 6.5-8.0 L-Carnitine 0.0-0.2 Taurine 0 05-0.5 


25. The formulation of claim 24, comprising ten or more of the recitedamino acids as modified release amino acids in the recited weight parts.26. The formulation of claim 24, comprising fifteen or more of therecited amino acids as modified release amino acids in the recitedweight parts.
 27. The formulation of claim 24, comprising all of therecited amino acids in the recited weight parts.
 28. The formulation ofclaim 24, comprising the recited large neutral amino acids as modifiedrelease amino acids in the recited weight parts.
 29. The formulation ofclaim 24, comprising the recited essential amino acids as modifiedrelease amino acids in the recited weight parts.
 30. The formulation ofclaim 24, comprising the recited brain chain amino acids as modifiedrelease amino acids in the recited weight parts.
 31. The formulation ofclaim 1, comprising five or more of the following amino acids asmodified release amino acids in the recited weight parts: Amino AcidWeight Parts L-Alanine 4.5-6.0 L-Arginine  8.5-10.5 L-Aspartic Acid7.0-8.5 L-Cystine 3.0-4.0 L-Glutamic Acid +  9.0-13.0 Glutamine Glycine7.3-9.5 L-Histidine 4.5-6.0 L-Isoleucine 0.0 L-Leucine 0.0 L-Lysine 8.5-10.5 L-Methionine 1.5-3.0 L-Phenylalanine 5.5-7.0 L-Proline 9.5-11.0 L-Serine 5.5-7.5 L-Threonine 6.5-8.0 L-Tryptophan 2.5-3.5L-Tyrosine 2.5-7.0 L-Valine 0.0 L-Carnitine 0.02-0.12 Taurine 0.05-1.0 


32. The formulation of claim 31, comprising ten or more of the recitedamino acids as modified release amino acids in the recited weight parts.33. The formulation of claim 31, comprising fifteen or more of therecited amino acids as modified release amino acids in the recitedweight parts.
 34. The formulation of claim 31, comprising all of therecited amino acids in the recited weight parts.
 35. The formulation ofclaim 31, comprising the recited large neutral amino acids as modifiedrelease amino acids in the recited weight parts.
 36. The formulation ofclaim 31, comprising the recited essential amino acids as modifiedrelease amino acids in the recited weight parts.
 37. The formulation ofclaim 31, comprising the recited brain chain amino acids as modifiedrelease amino acids in the recited weight parts.
 38. The formulation ofclaim 1, comprising five or more of the following amino acids asmodified release amino acids in the recited weight parts Amino AcidWeight Parts L-Alanine  9.0-11.0 L-Arginine  8.0-10.0 L-Aspartic Acid7.0-9.0 L-Cystine 3.0-4.0 L-Glutamic Acid +  9.0-11.0 Glutamine Glycine3.5-4.5 L-Histidine 5.5-7.0 L-Isoleucine 0.0-0.5 L-Leucine 12.0-15.0L-Lysine  8.0-10.5 L-Methionine 0.0 L-Phenylalanine 5.5-7.5 L-Proline3.5-5.0 L-Serine 5.0-6.5 L-Threonine 0.0 L-Tryptophan 2.0-3.5 L-Tyrosine5.0-7.0 L-Valine 0.0 L-Carnitine  0.0-0.15 Taurine 0.05-1.0 


39. The formulation of claim 38, comprising ten or more of the recitedamino acids as modified release amino acids in the recited weight parts.40. The formulation of claim 38, comprising fifteen or more of therecited amino acids as modified release amino acids in the recitedweight parts.
 41. The formulation of claim 38, comprising all of therecited amino acids in the recited weight parts.
 42. The formulation ofclaim 38, comprising the recited large neutral amino acids as modifiedrelease amino acids in the recited weight parts.
 43. The formulation ofclaim 38, comprising the recited essential amino acids as modifiedrelease amino acids in the recited weight parts.
 44. The formulation ofclaim 38, comprising the recited brain chain amino acids as modifiedrelease amino acids in the recited weight parts.
 45. The formulation ofclaim 1, comprising five or more of the following amino acids asmodified release amino acids in the recited weight parts: Amino AcidWeight Parts L-Alanine 4.0-5.0 L-Arginine 7.0-8.5 L-Aspartic Acid6.0-7.0 L-Cystine 2.5-3.5 L-Glutamic Acid +  8.0-10.0 Glutamine Glycine6.5-8.0 L-Histidine 3.5-5.5 L-Isoleucine 6.0-8.5 L-Leucine 10.5-13.0L-Lysine 0.0 L-Methionine 1.5-2.5 L-Phenylalanine 4.5-6.0 L-Proline7.5-9.0 L-Serine 4.5-6.0 L-Threonine 5.0-6.5 L-Tryptophan 0.0-0.5L-Tyrosine 4.5-6.0 L-Valine 6.5-8.0 L-Carnitine 0.0-0.2 Taurine 0.5-0.5


46. The formulation of claim 45, comprising ten or more of the recitedamino acids as modified release amino acids in the recited weight parts.47. The formulation of claim 45, comprising fifteen or more of therecited amino acids as modified release amino acids in the recitedweight parts.
 48. The formulation of claim 45, comprising all of therecited amino acids in the recited weight parts.
 49. The formulation ofclaim 45, comprising the recited large neutral amino acids as modifiedrelease amino acids in the recited weight parts.
 50. The formulation ofclaim 45, comprising the recited essential amino acids as modifiedrelease amino acids in the recited weight parts.
 51. The formulation ofclaim 45, comprising the recited brain chain amino acids as modifiedrelease amino acids in the recited weight parts.
 52. The formulation ofclaim 1, comprising five or more of the following amino acids asmodified release amino acids in the recited weight parts Amino AcidWeight Parts L-Alanine 4.0-6.0 L-Arginine 7.0-9.0 L-Aspartic Acid6.0-7.0 L-Cystine 2.5-3.5 L-Glutamic Acid + 8.0-9.5 Glutamine Glycine13.0-16.0 L-Histidine 4.0-5.0 L-Isoleucine 2.0-3.5 L-Leucine 0.0L-Lysine 6.0-7.5 L-Methionine 1.5-2.5 L-Phenylalanine 5.0-6.0 L-Proline13.0-15.0 L-Serine 4.0-5.0 L-Threonine 4.0-5.5 L-Tryptophan 1.5-2.5L-Tyrosine 4.5-6.0 L-Valine 2.5-4.0 L-Carnitine  0.0-0.15 Taurine0.05-0.5 


53. The formulation of claim 52, comprising ten or more of the recitedamino acids as modified release amino acids in the recited weight parts.54. The formulation of claim 52, comprising fifteen or more of therecited amino acids as modified release amino acids in the recitedweight parts.
 55. The formulation of claim 52, comprising all of therecited amino acids in the recited weight parts.
 56. The formulation ofclaim 52, comprising the recited large neutral amino acids as modifiedrelease amino acids in the recited weight parts.
 57. The formulation ofclaim 52, comprising the recited essential amino acids as modifiedrelease amino acids in the recited weight parts.
 58. The formulation ofclaim 52, comprising the recited brain chain amino acids as modifiedrelease amino acids in the recited weight parts.
 59. The formulation ofclaim 1, comprising five or more of the following amino acids asmodified release amino acids in the recited weight parts: Amino AcidWeight Parts L-Alanine 3.0-5.0 L-Arginine 6.5-8.0 L-Aspartic Acid5.0-7.0 L-Cystine 2.0-3.0 L-Glutamic Acid + 7.5-9.0 Glutamine Glycine5.5-7.0 L-Histidine 3.5-4.5 L-Isoleucine 5.5-7.0 L-Leucine 10.0-12.0L-Lysine  70-8.0 L-Methionine 0.0 L-Phenylalanine 4.0-5.5 L-Proline7.0-8.5 L-Serine 5.0-8.5 L-Threonine 5.0-6.0 L-Tryptophan 1.5-2.5L-Tyrosine 4.5-6.0 L-Valine 6.0-8.0 L-Carnitine 0.0-0.1 Taurine 0.5-0.5


60. The formulation of claim 59, comprising ten or more of the recitedamino acids as modified release amino acids in the recited weight parts.61. The formulation of claim 59, comprising fifteen or more of therecited amino acids as modified release amino acids in the recitedweight parts.
 62. The formulation of claim 59, comprising all of therecited amino acids in the recited weight parts.
 63. The formulation ofclaim 59, comprising the recited large neutral amino acids as modifiedrelease amino acids in the recited weight parts.
 64. The formulation ofclaim 59, comprising the recited essential amino acids as modifiedrelease amino acids in the recited weight parts.
 65. The formulation ofclaim 59, comprising the recited brain chain amino acids as modifiedrelease amino acids in the recited weight parts.
 66. The formulation ofclaim 1, comprising five or more of the following amino acids asmodified release amino acids in the recited weight parts Amino AcidWeight Parts L-Alanine 3.5-5.0 L-Arginine 6.5-8.5 L-Aspartic Acid6.0-8.0 L-Cystine 2.0-3.5 L-Glutamic Acid +  7.0-10.0 Glutamine Glycine0.0 L-Histidine 4.0-5.0 L-Isoleucine 6.0-8.5 L-Leucine 10.0-12.5L-Lysine 7.0-8.5 L-Methionine 1.0-2.5 L-Phenylalanine 4.5-6.0 L-Proline7.0-9.0 L-Serine 4.5-5.5 L-Threonine 5.0-6.5 L-Tryptophan 2.0-3.0L-Tyrosine 4.0-6.0 L-Valine 6.0-8.0 L-Carnitine 0.05-0.2  Taurine0.1-0.5


67. The formulation of claim 66, comprising ten or more of the recitedamino acids as modified release amino acids in the recited weight parts.68. The formulation of claim 66, comprising fifteen or more of therecited amino acids as modified release amino acids in the recitedweight parts.
 69. The formulation of claim 66, comprising all of therecited amino acids in the recited weight parts.
 70. The formulation ofclaim 66, comprising the recited large neutral amino acids as modifiedrelease amino acids in the recited weight parts.
 71. The formulation ofclaim 66, comprising the recited essential amino acids as modifiedrelease amino acids in the recited weight parts.
 72. The formulation ofclaim 66, comprising the recited brain chain amino acids as modifiedrelease amino acids in the recited weight parts.
 73. The formulation ofany of the foregoing claims wherein the formulation is a powder sachet,a liquid, or a chewable tablet.
 74. The formulation of any of theforegoing claims in the form of a tablet, a pill, a soft or hard gelatincapsules, a powder, a granulate, a microsphere, a lozenge, a sachet ofpackaged powders or granulates or microspheres, an elixir, a suspension,an emulsion, a solution, or a syrup.
 75. The formulation of any of theforegoing claim wherein the formulation comprises from 1 to 20 grams ofamino acids, wherein the formulation is therapeutically effective tomeet the amino acid dietary needs of a human patient when administeredthree to four times per day.
 76. The formulation of any of the foregoingclaims, where the formulation comprises from 25% to 75% essential aminoacids and from 25% to 75% non-essential amino acids based on the weightof the free base of all of the amino acids.
 77. The formulation of anyof the foregoing claims, wherein the formulation comprises a firstportion of the amino acids in an immediate release format and a secondportion of the amino acids in a modified release format.
 78. Theformulation of any of the foregoing claims wherein the essential aminoacids are in an immediate release format and the non-essential aminoacids are in a modified release format.
 79. The formulation of any ofthe foregoing claims wherein the non-essential amino acids are in animmediate release format and the essential amino acids are in a modifiedrelease format.
 80. The formulation of any of the foregoing claims,wherein the formulation further comprises one or more additionalingredients selected from the group consisting of vitamins, minerals andcarbohydrates.
 81. The formulation of any of the foregoing claims.wherein the formulation further comprises one or more additionalingredients selected from the group consisting of choline, inositol,vitamin A, vitamin D, vitamin E, vitamin K, vitamin C, thiamin,riboflavin, niacin, vitamin B6, folate, vitamin B12, biotin, pantothenicacid, potassium, calcium, magnesium, iron, zinc, copper, manganese,selenium, chromium, molybdenum, iodine, sodium, sulfur, phosphorus,docosahexaenoic acid, eicosapentaenoic acid, arachidonic acid, andlutein, and salts, chelates, esters and other derivatives thereof. 82.The formulation of any of the foregoing claims, wherein the formulationcomprises greater than 20 wt % amino acids.
 83. The formulation of anyof the foregoing claims wherein x % equals 70%.
 84. The formulation ofany of the foregoing claims wherein x % equals 60%.
 85. The formulationof any of the foregoing claims wherein x % equals 50%.
 86. Theformulation of any of the foregoing claims wherein the modified releaseamino acids are coated by from 5 wt % to 25 wt % of one or more releasemodifying excipients based on the weight of the amino acids.
 87. Theformulation of any of the foregoing claims wherein the modified releaseamino acids are granulates coated by from 5 wt % to 25 wt % of one ormore release modifying excipients based on the weight of the aminoacids.
 88. The formulation of any of the foregoing claims wherein themodified release amino acids are granulates coated by from 5 wt % to 25wt % of one or more release modifying excipients based on the weight ofthe amino acids, wherein the granulates comprise a plurality of modifiedrelease amino acids.
 89. The formulation of any of the foregoing claimswherein the modified release amino acids are granulates coated bycoating means for retarding the amino acid release rate.
 90. Theformulation of any of the foregoing claims wherein the modified releaseamino acids are granulates coated by coating means for achieving therecited release rate.
 91. The formulation of any of the foregoing claimswherein the modified release amino acids further comprise one or morerelease modifying excipients selected from the group consisting ofethylcellulose, glyceryl dibehenate, cellulose acetate, vinylacetate/vinyl chloride copolymers, acrylate/methacrylate copolymers,polyethylene oxide, hydroxypropyl methylcellulose, carrageenan, alginicacid and salts thereof, hydroxyethyl cellulose, hydroxypropyl cellulose,karaya gum, acacia gum, tragacanth gum, locust bean gum, guar gum,sodium carboxymethyl cellulose, methyl cellulose, beeswax, carnauba wax,cetyl alcohol, hydrogenated vegetable oils, stearyl alcohol, acrylicacid copolymers, sodium alginate, carrageenan, alginic acid, pectin,sodium carboxymethyl cellulose, or a combination thereof.
 92. Theformulation of claim 91, wherein the release modifying excipient ispresent as a coating on the amino acids in an amount of from 1 wt % to30 wt % based on the weight of the amino acids.
 93. The formulation ofany of the foregoing claims further comprising a bulking agent selectedfrom lactose, sucrose, dextrose, sorbitol, fructose, and cellulosepowder.
 94. The formulation of any of the foregoing claims furthercomprising a disintegrating agent selected from microcrystallinecellulose, starches, crospovidone, sodium starch glycolate, andcrosscarmellose sodium.
 95. The formulation of any of the foregoingclaims further comprising a glidant or lubricant selected from talc,corn starch, silicon dioxide, sodium lauryl sulfate, magnesium stearate,calcium stearate, sodium stearate, stearic acid, sodium stearylfumarate, hydrogenated cotton seed oil, talc, waxes, cetyl alcohol,glyceryl stearate, glyceryl palmitate, glyceryl behenate, hydrogenatedvegetable oils, and stearyl alcohol.
 96. The formulation of any of theforegoing claims further comprising a binder selected from polyvinylpyrrollidone, starch, methylcellulose, hydroxypropyl methylcellulose,carboxymethyl cellulose, sucrose solution, dextrose solution, guar gum,xanthan gum, acacia, tragacanth, locust bean gum and sodium alginate, oran alginic acid salt.
 97. The formulation of any of the foregoing claimsfurther comprising a taste-masking agent selected from cellulosehydroxypropyl ethers (HPC); low-substituted hydroxypropyl ethers(L-HPC); cellulose hydroxypropyl methyl ethers (HPMC); methylcellulosepolymers; Ethylcelluloses (EC) and mixtures thereof; Polyvinyl alcohol(PVA); hydroxyethylcelluloses; carboxymethylcelluloses and salts ofcarboxymethylcelluloses (CMC); polyvinyl alcohol and polyethylene glycolco-polymers; monoglycerides, triglycerides, polyethylene glycols,modified food starch, acrylic polymers and mixtures of acrylic polymerswith cellulose ethers; cellulose acetate phthalate; sepifilms such asmixtures of HPMC and stearic acid, cyclodextrins, and mixtures thereof.98. The formulation of any of the foregoing claims comprising aflavoring agent selected from acacia syrup, acesulfame K, alitame,anise, apple, aspartame, banana, Bavarian cream, berry, black currant,butterscotch, calcium citrate, camphor, caramel, cherry, cherry cream,chocolate, cinnamon, bubble gum, citrus, citrus punch, citrus cream,cotton candy, cocoa, cola, cool cherry, cool citrus, cyclamate,cylamate, dextrose, eucalyptus, eugenol, fructose, fruit punch, ginger,glycyrrhetinate, glycyrrhiza (licorice) syrup, grape, grapefruit, honey,isomalt, lemon, lime, lemon cream, monoammonium glyrrhizinate, maltol,mannitol, maple, marshmallow, menthol, mint cream, mixed berry,neohesperidine DC, neotame, orange, pear, peach, peppermint, peppermintcream, raspberry, root beer, rum, saccharin, safrole, sorbitol,spearmint, spearmint cream, strawberry, strawberry cream, stevia,sucralose, sucrose, sodium saccharin, saccharin, aspartame, neotame,acesulfame potassium, mannitol, talin, xylitol, sucralose, sorbitol,swiss cream, tagatose, tangerine, thaumatin, tutti fruitti, vanilla,walnut, watermelon, wild cherry, wintergreen, xylitol, or a combinationthereof.
 99. The formulation of any of the foregoing claims made by aprocess selected from microencapsulation, spray drying, spinning disk,hot melt, spray chilling, fluidized bed, electrostatic deposition,centrifugal extrusion, rotational suspension separation, polymerizationat liquid-gas or solid-gas interface, pressure extrusion, or sprayingsolvent extraction bath, complex coacervation, solvent evaporation,polymer-polymer incompatibility, interfacial polymerization in liquidmedia, in situ polymerization, in-liquid drying, and desolvation inliquid media, dry granulation, extrusion and spheronization.
 100. Theformulation of any of the foregoing claims, wherein tyrosine in theformulation is not modified release.
 101. A method of supplementingamino acids to a mammalian subject in need thereof comprising orallyadministering to the subject a formulation defined by any of theforegoing claims.
 102. A method of treating a metabolic disorder in apatient in need thereof selected from the group consisting ofphenylketonuria, tyrosinemia, leucinosis, methylmalonic acidemia,homocystinuria, hyperglycinemia, isovaleric acidemia, propionicacidemia, and glutamic acidemia, in a patient in need thereof,comprising orally administering to the patient a formulation defined byany of the foregoing claims.
 103. The method of claim 101 or 102,wherein the formulation comprises a therapeutically effective amount ofamino acids when administered three or four times daily.
 104. A methodof making a formulation of any of the foregoing claims comprising: a.providing a first mixture comprising a plurality of amino acids; b.contacting the mixture with a wetting agent and a binder to form a wetmixture; c. optionally passing the wet mixture through a sieve to form auniform wet granulate; d. drying the uniform wet granulate to form a drygranulate; e. optionally passing the dry granulate through a sieve toform a uniform dry granulate; and f. optionally coating the uniform drygranulate with a modified release composition.
 105. The method of claim104, further comprising compressing the uniform dry granulate into achewable tablet.